Liquid pharmaceutical formulations for injection comprising procaine and uses thereof

ABSTRACT

Concentrated, low-viscosity, low-volume liquid pharmaceutical formulations of proteins have been developed. Such formulations can be rapidly and conveniently administered by subcutaneous or intramuscular injection, rather than by lengthy intravenous infusion. These formulations include low-molecular-weight and/or high-molecular-weight proteins, such as mAbs, and viscosity-lowering agents that are typically bulky polar organic compounds, such as many of the GRAS (US Food and Drug Administration List of compounds generally regarded as safe) and inactive injectable ingredients and FDA approved therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/030,521, filed Jul. 29, 2014, entitled “Low-ViscosityProtein Formulations Containing Hydrophobic Salts;” U.S. ProvisionalApplication No. 62/026,497, filed Jul. 18, 2014, entitled “Low-ViscosityProtein Formulations Containing GRAS Viscosity-Reducing Agents;” U.S.Provisional Application No. 62/008,050, filed Jun. 5, 2014, entitled“Low-Viscosity Protein Formulations Containing Ionic Liquids;” U.S.Provisional Application No. 61/988,005, filed May 2, 2014, entitled“Low-Viscosity Protein Formulations Containing Organophosphates;” U.S.Provisional Application No. 61/946,436, filed Feb. 28, 2014, entitled“Concentrated, Low-Viscosity Infliximab Formulations;” U.S. ProvisionalApplication No. 61/943,197, filed Feb. 21, 2014, entitled “Concentrated,Low-Viscosity, High-Molecular-Weight-Protein Formulations;” U.S.Provisional Application No. 61/940,227, filed Feb. 14, 2014, entitled“Concentrated, Low-Viscosity High-Molecular-Weight ProteinFormulations;” and U.S. Provisional Application No. 61,876,621, filedSep. 11, 2013, entitled “Concentrated, Low-Viscosity,High-Molecular-Weight Protein Formulations,” the disclosures of whichare expressly incorporated hereby by reference.

FIELD OF THE INVENTION

The invention is generally in the field of injectable low-viscositypharmaceutical formulations of highly concentrated proteins and methodsof making and using thereof.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (mAbs) are important protein-based therapeuticsfor treating various human diseases such as cancer, infectious diseases,inflammation, and autoimmune diseases. More than 20 mAb products havebeen approved by the U.S. Food and Drug Administration (FDA), andapproximately 20% of all biopharmaceuticals currently being evaluated inclinical trials are mAbs (Daugherty et al., Adv. Drug Deliv. Rev.58:686-706, 2006; and Buss et al., Curr. Opinion in Pharmacol.12:615-622, 2012).

mAb-based therapies are usually administered repeatedly over an extendedperiod of time and require several mg/kg dosing. Antibody solutions orsuspensions can be administered via parenteral routes, such as byintravenous (IV) infusions, and subcutaneous (SC) or intramuscular (IM)injections. The SC or IM routes reduce the treatment cost, increasepatient compliance, and improve convenience for patients and healthcareproviders during administration compared to the IV route. To beeffective and pharmaceutically acceptable, parenteral formulationsshould preferably be sterile, stable, injectable (e.g., via a syringe),and non-irritating at the site of injection, in compliance with FDAguidelines. Because of the small volumes required for subcutaneous(usually under about 2 mL) and intramuscular (usually under about 5 mL)injections, these routes of administration for high-dose proteintherapies require concentrated protein solutions. These highconcentrations often result in very viscous formulations that aredifficult to administer by injection, cause pain at the site ofinjection, are often imprecise, and/or may have decreased chemicaland/or physical stability.

These characteristics result in manufacturing, storage, and usagerequirements that can be challenging to achieve, in particular forformulations having high concentrations of high-molecular-weightproteins, such as mAbs. All protein therapeutics to some extent aresubject to physical and chemical instability, such as aggregation,denaturation, crosslinking, deamidation, isomerization, oxidation, andclipping (Wang et al., J. Pharm. Sci. 96:1-26, 2007). Thus, optimalformulation development is paramount in the development of commerciallyviable protein pharmaceuticals.

High protein concentrations pose challenges relating to the physical andchemical stability of the protein, as well as difficulty withmanufacture, storage, and delivery of the protein formulation. Oneproblem is the tendency of proteins to aggregate and form particulatesduring processing and/or storage, which makes manipulations duringfurther processing and/or delivery difficult. Concentration-dependentdegradation and/or aggregation are major challenges in developingprotein formulations at higher concentrations. In addition to thepotential for non-native protein aggregation and particulate formation,reversible self-association in aqueous solutions may occur, whichcontributes to, among other things, increased viscosity that complicatesdelivery by injection. (See, for example, Steven J. Shire et al., J.Pharm. Sci. 93:1390-1402, 2004.) Increased viscosity is one of the keychallenges encountered in concentrated protein compositions affectingboth production processes and the ability to readily deliver suchcompositions by conventional means. (See, for example, J. Jezek et al.,Advanced Drug Delivery Reviews 63:1107-1117, 2011.)

Highly viscous liquid formulations are difficult to manufacture, drawinto a syringe, and inject subcutaneously or intramuscularly. The use offorce in manipulating the viscous formulations can lead to excessivefrothing, which may further denature and inactivate the therapeuticallyactive protein. High viscosity solutions also require larger diameterneedles for injection and produce more pain at the injection site.

Currently available commercial mAb products administered by SC or IMinjection are usually formulated in aqueous buffers, such as a phosphateor L-histidine buffer, with excipients or surfactants, such as mannitol,sucrose, lactose, trehalose, POLOXAMER® (nonionic triblock copolymerscomposed of a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide))) or POLYSORBATE® 80(PEG(80)sorbitan monolaurate), to prevent aggregation and improvestability. Reported antibody concentrations formulated as describedabove are typically up to about 100 mg/mL (Wang et al., J. Pharm. Sci.96:1-26, 2007).

U.S. Pat. No. 7,758,860 describes reducing the viscosity in formulationsof low-molecular-weight proteins using a buffer and a viscosity-reducinginorganic salt, such as calcium chloride or magnesium chloride. Thesesame salts, however, showed little effect on the viscosity of ahigh-molecular-weight antibody (IMA-638) formulation. As described inU.S. Pat. No. 7,666,413, the viscosity of aqueous formulations ofhigh-molecular-weight proteins has been reduced by the addition of suchsalts as arginine hydrochloride, sodium thiocyanate, ammoniumthiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zincchloride, or sodium acetate in a concentration of greater than about 100mM or, as described in U.S. Pat. No. 7,740,842, by addition of organicor inorganic acids. However, these salts do not reduce the viscosity toa desired level and in some cases make the formulation so acidic that itis likely to cause pain at the site of injection.

U.S. Pat. No. 7,666,413 describes reduced-viscosity formulationscontaining specific salts and a reconstituted anti-IgE mAb, but with amaximum antibody concentration of only up to about 140 mg/mL. U.S. Pat.No. 7,740,842 describes E25 anti-IgE mAb formulations containingacetate/acetic acid buffer with antibody concentrations up to 257 mg/mL.The addition of salts such as NaCl, CaCl₂, or MgCl₂ was demonstrated todecrease the dynamic viscosity under high-shear conditions; however, atlow-shear the salts produced an undesirable and dramatic increase in thedynamic viscosity. Additionally, inorganic salts such as NaCl may lowersolution viscosity and/or decrease aggregation (EP 1981824).

Non-aqueous antibody or protein formulations have also been described.WO2006/071693 describes a non-aqueous suspension of up to 100 mg/mL mAbin a formulation having a viscosity enhancer (polyvinylpyrrolidone, PVP)and a solvent (benzyl benzoate or PEG 400). WO2004/089335 describes 100mg/mL non-aqueous lysozyme suspension formulations containing PVP,glycofurol, benzyl benzoate, benzyl alcohol, or PEG 400.US2008/0226689A1 describes 100 mg/mL human growth hormone (hGH) singlephase, three vehicle component (polymer, surfactant, and a solvent),non-aqueous, viscous formulations. U.S. Pat. No. 6,730,328 describesnon-aqueous, hydrophobic, non-polar vehicles of low reactivity, such asperfluorodecalin, for protein formulations. These formulations arenon-optimal and have high viscosities that impair processing,manufacturing and injection; lead to the presence of multiple vehiclecomponents in the formulations; and present potential regulatorychallenges associated with using polymers not yet approved by the FDA.

Alternative non-aqueous protein or antibody formulations have beendescribed using organic solvents, such as benzyl benzoate (Miller etal., Langmuir 26:1067-1074, 2010), benzyl acetate, ethanol, or methylethyl ketone (Srinivasan et al., Pharm. Res. 30:1749-1757, 2013). Inboth instances, viscosities of less than 50 centipoise (cP) wereachieved upon formulation at protein concentrations of at least about200 mg/mL. U.S. Pat. No. 6,252,055 describes mAb formulations withconcentrations ranging from 100 mg/mL up to 257 mg/mL. Formulations withconcentrations greater than about 189 mg/mL demonstrated dramaticallyincreased viscosities, low recovery rates, and difficulty in processing.U.S. Patent Application Publication No. 2012/0230982 describes antibodyformulations with concentrations of 100 mg/mL to 200 mg/mL. None ofthese formulations are low enough viscosity for ease of injection.

Du and Klibanov (Biotechnology and Bioengineering 108:632-636, 2011)described reduced viscosity of concentrated aqueous solutions of bovineserum albumin with a maximum concentration up to 400 mg/mL and bovinegamma globulin with a maximum concentration up to 300 mg/mL. Guo et al.(Pharmaceutical Research 29:3102-3109, 2012) described low-viscosityaqueous solutions of four model mAbs achieved using hydrophobic salts.The mAb formulation employed by Guo had an initial viscosity, prior toadding salts, no greater than 73 cP. The viscosities of manypharmaceutically important mAbs, on the other hand, can exceed 1,000 cPat therapeutically relevant concentrations.

It is not a trivial matter to control aggregation and viscosity inhigh-concentration mAb solutions (EP 2538973). This is evidenced by thefew mAb products currently on the market as high-concentrationformulations (>100 mg/mL) (EP 2538973).

The references cited above demonstrate that while many groups haveattempted to prepare low-viscosity formulations of mAbs and othertherapeutically important proteins, a truly useful formulation for manyproteins has not yet been achieved. Notably, many of the above reportsemploy agents for which safety and toxicity profiles have not been fullyestablished. These formulations would therefore face a higher regulatoryburden prior to approval than formulations containing compounds known tobe safe. Indeed, even if a compound were to be shown to substantiallyreduce viscosity, the compound may ultimately be unsuitable for use in aformulation intended for injection into a human.

Many pharmaceutically important high-molecular-weight proteins, such asmAbs, are currently administered via IV infusions in order to delivertherapeutically effective amounts of protein due to problems with highviscosity and other properties of concentrated solutions of largeproteins. For example, to provide a therapeutically effective amount ofmany high-molecular-weight proteins, such as mAbs, in volumes less thanabout 2 mL, protein concentrations greater than 150 mg/mL are oftenrequired.

It is, therefore, an object of the present invention to provideconcentrated, low-viscosity liquid formulations of pharmaceuticallyimportant proteins, especially high-molecular-weight proteins, such asmAbs.

It is a further object of the present invention to provide concentratedlow-viscosity liquid formulations of proteins, especiallyhigh-molecular-weight proteins, such as mAbs, capable of deliveringtherapeutically effective amounts of these proteins in volumes usefulfor SC and IM injections.

It is a further object of the present invention to provide theconcentrated liquid formulations of proteins, especiallyhigh-molecular-weight proteins, such as mAbs, with low viscosities thatcan improve injectability and/or patient compliance, convenience, andcomfort.

It is also an object of the present invention to provide methods formaking and storing concentrated, low-viscosity formulations of proteins,especially high-molecular-weight proteins, such as mAbs.

It is an additional object of the present invention to provide methodsof administering low-viscosity, concentrated liquid formulations ofproteins, especially high-molecular-weight proteins, such as mAbs. It isan additional object of the present invention to provide methods forprocessing reduced-viscosity, high-concentration biologics withconcentration and filtration techniques known to those skilled in theart.

SUMMARY OF THE INVENTION

Concentrated, low-viscosity, low-volume liquid pharmaceuticalformulations of proteins have been developed. Such formulations can berapidly and conveniently administered by subcutaneous (SC) orintramuscular (IM) injection, rather than by lengthy intravenousinfusion. These formulations include low-molecular-weight and/orhigh-molecular-weight proteins, such as mAbs, and viscosity-loweringagents that are typically bulky polar organic compounds, such as many ofthe GRAS (US Food and Drug Administration's list of compounds generallyregarded as safe), inactive injectable ingredients and FDA-approvedtherapeutics.

The concentration of proteins is between about 10 mg/mL and about 5,000mg/mL, more preferably from about 100 mg/mL to about 2,000 mg/mL. Insome embodiments, the concentration of proteins is between about 100mg/mL to about 500 mg/mL, more preferably from about 300 mg/mL to about500 mg/mL. Formulations containing proteins and viscosity-loweringagents are stable when stored at a temperature of 4° C., for a period ofat least one month, preferably at least two months, and most preferablyat least three months. The viscosity of the formulation is less thanabout 75 cP, preferably below 50 cP, and most preferably below 20 cP atabout 25° C. In some embodiments, the viscosity is less than about 15 cPor even less than or about 10 cP at about 25° C. In certain embodiments,the viscosity of the formulation is about 10 cP. Formulations containingproteins and viscosity-lowering agents typically are measured at shearrates from about 0.6 s⁻¹ to about 450 s⁻¹, and preferably from about 2s⁻¹ to about 400 s⁻¹, when measured using a cone and plate viscometer.Formulations containing proteins and viscosity-lowering agents typicallyare measured at shear rates from about 3 s⁻¹ to about 55,000 s⁻¹, andpreferably from about 20 s⁻¹ to about 2,000 s⁻¹, when measured using amicrofluidic viscometer.

The viscosity of the protein formulation is reduced by the presence ofone or more viscosity-lowering agents. Unless specifically statedotherwise, the term “viscosity-lowering agent” includes both singlecompounds and mixtures of two or more compounds. It is preferred thatthe viscosity-lowering agent is present in the formulation at aconcentration less than about 1.0 M, preferably less than about 0.50 M,more preferably less than about 0.30 M, and most preferably less thanabout 0.15 M. In some embodiments, the viscosity-lowering agent ispresent in the formulation in concentrations as low as 0.01 M. Theformulations can have a viscosity that is at least about 30% less,preferably at least about 50% less, most preferably at least about 75%less, than the viscosity of the corresponding formulation under the sameconditions except for replacement of the viscosity-lowering agent withan appropriate buffer or salt of about the same concentration. In someembodiments, a low-viscosity formulation is provided where the viscosityof the corresponding formulation without the viscosity-lowering agent isgreater than about 200 cP, greater than about 500 cP, or even aboveabout 1,000 cP. In a preferred embodiment, the shear rate of theformulation is at least about 0.5 s⁻¹, when measured using a cone andplate viscometer or at least about 1.0 s⁻¹, when measured using amicrofluidic viscometer.

For embodiments in which the protein is a “high-molecular-weightprotein”, the high molecular weight protein may have a molecular weightbetween about 100 kDa and about 1,000 kDa, preferably between about 120kDa and about 500 kDa, and most preferably between about 120 kDa andabout 250 kDa. The high-molecular-weight protein can be an antibody,such as a mAb, or a PEGylated, or otherwise a derivatized form thereof.Preferred mAbs include natalizumab (TYSABRI®), cetuximab (ERBITUX®),bevacizumab (AVASTIN®), trastuzumab) (HERCEPTIN®, infliximab(REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab(ARZERRA®), and biosimilars thereof. The high-molecular-weight protein,optionally PEGylated, can be an enzyme. Other proteins and mixtures ofproteins may also be formulated to reduce their viscosity.

In some embodiments, the protein and viscosity-lowering agent areprovided in a lyophilized dosage unit, sized for reconstitution with asterile aqueous pharmaceutically acceptable vehicle, to yield theconcentrated low-viscosity liquid formulations. The presence of theviscosity-lowering agent(s) facilitates and/or accelerates thereconstitution of the lyophilized dosage unit compared to a lyophilizeddosage unit not containing a viscosity-lowering agent.

Methods are provided herein for preparing concentrated, low-viscosityliquid formulations of high-molecular-weight proteins such as mAbs, aswell as methods for storing the low-viscosity, high-concentrationprotein formulations, and for administration thereof to patients. Inanother embodiment, the viscosity-lowering agent is added to facilitateprocessing (e.g., pumping, concentration, and/or filtration) by reducingthe viscosity of the protein solutions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the viscosity in cP as a function of the proteinconcentration (in mg/mL) for solutions of biosimilar cetuximab(ERBITUX®) in 0.25 M phosphate buffer (PB; diamonds) and a solutioncontaining 0.25 M camphorsulfonic acid L-lysine (CSAL; squares) at 25°C. and final pH of 7.0. The data points incorporate standard deviationswhich, however, are often smaller than the symbols.

FIGS. 2A and 2B depict the viscosity in cP as a function of the proteinconcentration (in mg/mL) for solutions of biosimilar bevacizumab(AVASTIN®) in 0.25 M phosphate buffer (PB; diamonds) and 0.25 M CSAL(squares) at 25° C. and final pH of 7.0. The data points incorporatestandard deviations which, however, are often smaller than the symbols.

FIG. 3 is a graph of the viscosity (cP) of aqueous solutions of 200±9mg/mL biosimilar bevacizumab (AVASTIN®) as a function of pH along thex-axis containing either phosphate-citrate buffer or camphorsulfonicacid arginine (CSAA) at a concentration of 0.25 M.

FIG. 4 is a bar graph comparing the fold reduction in viscosity as afunction of pH for aqueous solutions containing biosimilar bevacizumab(AVASTIN®; at approximately 200 mg/mL or 226 mg/mL) and 0.25 Mcamphorsulfonic acid arginine (CSAA). The fold reduction is computed asthe ratio of the viscosity (cP) in phosphate-citrate buffer to theviscosity (cP) in the 0.25 M CSAA solution.

FIG. 5 is a graph of the viscosity (cP) of aqueous solutions ofbiosimilar cetuximab (ERBITUX®; at 202±5 mg/mL, 229±5 mg/mL, or 253±4mg/mL) containing 0.25 M CSAA as a function of pH along the x-axis at25° C.

FIGS. 6A and 6B are size-exclusion chromatography traces depictingabsorbance intensity (at 280 nm) as a function of elution time (inminutes) for a 220 mg/mL aqueous solution of REMICADE® stored at 4° C.for up to 100 days, compared to freshly reconstituted commercial drugproduct.

FIG. 7 depicts the viscosity (cP) as a function of protein concentration(mg/mL) of aqueous solutions of biosimilar bevacizumab (AVASTIN®) in0.25 M phosphate buffer, 0.10 M or 0.25 M APMI*2HCl((1-(3-aminopropyl)-2-methyl-1H-imidazole bis-HCl).

FIG. 8 depicts the viscosity (cP) as a function of protein concentration(mg/mL) of aqueous solutions of biosimilar bevacizumab (AVASTIN®) in0.25 M phosphate buffer, 0.10 M thiamine pyrophosphate (TPP), or 0.10 MTPP1-(3-aminopropyl)-2-methyl-1H-imidazole (APMI).

FIG. 9 depicts the viscosity (cP) of aqueous solutions of golimumab(SIMPONI ARIA®) as a function of protein concentration (mg/mL) with 0.15M phosphate buffer or 0.15 M thiamine HCl.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “protein,” as generally used herein, refers to a polymer ofamino acids linked to each other by peptide bonds to form a polypeptidefor which the chain length is sufficient to produce at least adetectable tertiary structure. Proteins having a molecular weight(expressed in kDa wherein “Da” stands for “Daltons” and 1 kDa=1,000 Da)greater than about 100 kDa may be designated “high-molecular-weightproteins,” whereas proteins having a molecular weight less than about100 kDa may be designated “low-molecular-weight proteins.” The term“low-molecular-weight protein” excludes small peptides lacking therequisite of at least tertiary structure necessary to be considered aprotein. Protein molecular weight may be determined using standardmethods known to one skilled in the art, including, but not limited to,mass spectrometry (e.g., ESI, MALDI) or calculation from known aminoacid sequences and glycosylation. Proteins can be naturally occurring ornon-naturally occurring, synthetic, or semi-synthetic.

“Essentially pure protein(s)” and “substantially pure protein(s)” areused interchangeably herein and refer to a composition comprising atleast about 90% by weight pure protein, preferably at least about 95%pure protein by weight. “Essentially homogeneous” and “substantiallyhomogeneous” are used interchangeably herein and refer to a compositionwherein at least about 90% by weight of the protein present is acombination of the monomer and reversible di- and oligo-meric associates(not irreversible aggregates), preferably at least about 95%.

The term “antibody,” as generally used herein, broadly covers mAbs(including full-length antibodies which have an immunoglobulin Fcregion), antibody compositions with polyepitopic specificity, bispecificantibodies, diabodies, and single-chain antibody molecules, as well asantibody fragments (e.g., Fab, Fab′, F(ab′)₂, and Fv), single domainantibodies, multivalent single domain antibodies, Fab fusion proteins,and fusions thereof.

The term “monoclonal antibody” or “mAb,” as generally used herein,refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical, except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single epitope. These aretypically synthesized by culturing hybridoma cells, as described byKohler et al. (Nature 256: 495, 1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), or isolated from phageantibody libraries using the techniques described in Clackson et al.(Nature 352: 624-628, 1991) and Marks et al. (J. Mol. Biol. 222:581-597, 1991), for example. As used herein, “mAbs” specifically includederivatized antibodies, antibody-drug conjugates, and “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is (are)identical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such antibodies, so long as theyexhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

An “antibody fragment” comprises a portion of an intact antibody,including the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870;Zapata et al., Protein Eng. 8:1057-1062, 1995); single-chain antibodymolecules; multivalent single domain antibodies; and multispecificantibodies formed from antibody fragments.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences ofantibodies) of mostly human sequences, which contain minimal sequencesderived from non-human immunoglobulin. (See, e.g., Jones et al., Nature321:522-525, 1986; Reichmann et al., Nature 332:323-329, 1988; andPresta, Curr. Op. Struct. Biol. 2:593-596, 1992.)

“Rheology” refers to the study of the deformation and flow of matter.

“Viscosity” refers to the resistance of a substance (typically a liquid)to flow. Viscosity is related to the concept of shear force; it can beunderstood as the effect of different layers of the fluid exertingshearing force on each other, or on other surfaces, as they move againsteach other. There are several measures of viscosity. The units ofviscosity are Ns/m², known as Pascal-seconds (Pa-s). Viscosity can be“kinematic” or “absolute”. Kinematic viscosity is a measure of the rateat which momentum is transferred through a fluid. It is measured inStokes (St). The kinematic viscosity is a measure of the resistive flowof a fluid under the influence of gravity. When two fluids of equalvolume and differing viscosity are placed in identical capillaryviscometers and allowed to flow by gravity, the more viscous fluid takeslonger than the less viscous fluid to flow through the capillary. If,for example, one fluid takes 200 seconds (s) to complete its flow andanother fluid takes 400 s, the second fluid is called twice as viscousas the first on a kinematic viscosity scale. The dimension of kinematicviscosity is length²/time. Commonly, kinematic viscosity is expressed incentiStokes (cSt). The SI unit of kinematic viscosity is mm²/s, which isequal to 1 cSt. The “absolute viscosity,” sometimes called “dynamicviscosity” or “simple viscosity,” is the product of kinematic viscosityand fluid density. Absolute viscosity is expressed in units ofcentipoise (cP). The SI unit of absolute viscosity is themilliPascal-second (mPa-s), where 1 cP=1 mPa-s.

Viscosity may be measured by using, for example, a viscometer at a givenshear rate or multiple shear rates. An “extrapolated zero-shear”viscosity can be determined by creating a best fit line of the fourhighest-shear points on a plot of absolute viscosity versus shear rate,and linearly extrapolating viscosity back to zero-shear. Alternatively,for a Newtonian fluid, viscosity can be determined by averagingviscosity values at multiple shear rates. Viscosity can also be measuredusing a microfluidic viscometer at single or multiple shear rates (alsocalled flow rates), wherein absolute viscosity is derived from a changein pressure as a liquid flows through a channel. Viscosity equals shearstress over shear rate. Viscosities measured with microfluidicviscometers can, in some embodiments, be directly compared toextrapolated zero-shear viscosities, for example those extrapolated fromviscosities measured at multiple shear rates using a cone and plateviscometer.

“Shear rate” refers to the rate of change of velocity at which one layerof fluid passes over an adjacent layer. The velocity gradient is therate of change of velocity with distance from the plates. This simplecase shows the uniform velocity gradient with shear rate (v₁−v₂)/h inunits of (cm/sec)/(cm)=1/sec. Hence, shear rate units are reciprocalseconds or, in general, reciprocal time. For a microfluidic viscometer,change in pressure and flow rate are related to shear rate. “Shear rate”is to the speed with which a material is deformed. Formulationscontaining proteins and viscosity-lowering agents are typically measuredat shear rates ranging from about 0.5 s⁻¹ to about 200 s⁻¹ when measuredusing a cone and plate viscometer and a spindle appropriately chosen byone skilled in the art to accurately measure viscosities in theviscosity range of the sample of interest (i.e., a sample of 20 cP ismost accurately measured on a CPE40 spindle affixed to a DV2T viscometer(Brookfield)); greater than about 20 s⁻¹ to about 3,000 s⁻¹ whenmeasured using a microfluidic viscometer.

For classical “Newtonian” fluids, as generally used herein, viscosity isessentially independent of shear rate. For “non-Newtonian fluids,”however, viscosity either decreases or increases with increasing shearrate, e.g., the fluids are “shear thinning” or “shear thickening”,respectively. In the case of concentrated (i.e., high-concentration)protein solutions, this may manifest as pseudoplastic shear-thinningbehavior, i.e., a decrease in viscosity with shear rate.

The term “chemical stability,” as generally used herein, refers to theability of the protein components in a formulation to resist degradationvia chemical pathways, such as oxidation, deamidation, or hydrolysis. Aprotein formulation is typically considered chemically stable if lessthan about 5% of the components are degraded after 24 months at 4° C.

The term “physical stability,” as generally used herein, refers to theability of a protein formulation to resist physical deterioration, suchas aggregation. A formulation that is physically stable forms only anacceptable percentage of irreversible aggregates (e.g., dimers, trimers,or other aggregates) of the bioactive protein agent. The presence ofaggregates may be assessed in a number of ways, including by measuringthe average particle size of the proteins in the formulation by means ofdynamic light scattering. A formulation is considered physically stableif less than about 5% irreversible aggregates are formed after 24 monthsat 4° C. Acceptable levels of aggregated contaminants ideally would beless than about 2%. Levels as low as about 0.2% are achievable, althoughapproximately 1% is more typical.

The term “stable formulation,” as generally used herein, means that aformulation is both chemically stable and physically stable. A stableformulation may be one in which more than about 95% of the bioactiveprotein molecules retain bioactivity in a formulation after 24 months ofstorage at 4° C., or equivalent solution conditions at an elevatedtemperature, such as one month storage at 40° C. Various analyticaltechniques for measuring protein stability are available in the art andare reviewed, for example, in Peptide and Protein Drug Delivery,247-301, Vincent Lee, Ed., Marcel Dekker, Inc., New York, N.Y. (1991)and Jones, A., Adv. Drug Delivery Revs. 10:29-90, 1993. Stability can bemeasured at a selected temperature for a certain time period. For rapidscreening, for example, the formulation may be kept at 40° C., for 2weeks to one month, at which time residual biological activity ismeasured and compared to the initial condition to assess stability. Whenthe formulation is to be stored at 2° C.-8° C., generally theformulation should be stable at 30° C. or 40° C. for at least one monthand/or stable at 2° C.-8° C. for at least 2 years. When the formulationis to be stored at room temperature, about 25° C., generally theformulation should be stable for at least 2 years at about 25° C. and/orstable at 40° C. for at least about 6 months. The extent of aggregationfollowing lyophilization and storage can be used as an indicator ofprotein stability. In some embodiments, the stability is assessed bymeasuring the particle size of the proteins in the formulation. In someembodiments, stability may be assessed by measuring the activity of aformulation using standard biological activity or binding assays wellwithin the abilities of one ordinarily skilled in the art.

The term protein “particle size,” as generally used herein, means theaverage diameter of the predominant population of bioactive moleculeparticulates, or particle size distributions thereof, in a formulationas determined by using well known particle sizing instruments, forexample, dynamic light scattering, SEC (size exclusion chromatography),or other methods known to one ordinarily skilled in the art.

The term “concentrated” or “high-concentration”, as generally usedherein, describes liquid formulations having a final concentration ofprotein greater than about 10 mg/mL, preferably greater than about 50mg/mL, more preferably greater than about 100 mg/mL, still morepreferably greater than about 200 mg/mL, or most preferably greater thanabout 250 mg/mL.

A “reconstituted formulation,” as generally used herein, refers to aformulation which has been prepared by dissolving a dry powder,lyophilized, spray-dried or solvent-precipitated protein in a diluent,such that the protein is dissolved or dispersed in aqueous solution foradministration.

A “lyoprotectant” is a substance which, when combined with a protein,significantly reduces chemical and/or physical instability of theprotein upon lyophilization and/or subsequent storage. Exemplarylyoprotectants include sugars and their corresponding sugar alcohols,such as sucrose, lactose, trehalose, dextran, erythritol, arabitol,xylitol, sorbitol, and mannitol; amino acids, such as arginine orhistidine; lyotropic salts, such as magnesium sulfate; polyols, such aspropylene glycol, glycerol, poly(ethylene glycol), or polypropyleneglycol); and combinations thereof. Additional exemplary lyoprotectantsinclude gelatin, dextrins, modified starch, and carboxymethyl cellulose.Preferred sugar alcohols are those compounds obtained by reduction ofmono- and di-saccharides, such as lactose, trehalose, maltose,lactulose, and maltulose. Additional examples of sugar alcohols areglucitol, maltitol, lactitol and isomaltulose. The lyoprotectant isgenerally added to the pre-lyophilized formulation in a “lyoprotectingamount.” This means that, following lyophilization of the protein in thepresence of the lyoprotecting amount of the lyoprotectant, the proteinessentially retains its physical and chemical stability and integrity.

A “diluent” or “carrier,” as generally used herein, is apharmaceutically acceptable (i.e., safe and non-toxic for administrationto a human or another mammal) and useful ingredient for the preparationof a liquid formulation, such as an aqueous formulation reconstitutedafter lyophilization. Exemplary diluents include sterile water,bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution, and combinations thereof.

A “preservative” is a compound which can be added to the formulationsherein to reduce contamination by and/or action of bacteria, fungi, oranother infectious agent. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation. Examples of potential preservatives includeoctadecyldimethylbenzylammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chained), and benzethoniumchloride. Other types of preservatives include aromatic alcohols such asphenol, butyl and benzyl alcohol, alkyl parabens such as methyl orpropyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, andm-cresol.

A “bulking agent,” as generally used herein, is a compound which addsmass to a lyophilized mixture and contributes to the physical structureof the lyophilized cake (e.g. facilitates the production of anessentially uniform lyophilized cake which maintains an open porestructure). Exemplary bulking agents include mannitol, glycine, lactose,modified starch, poly(ethylene glycol), and sorbitol.

A “therapeutically effective amount” is the least concentration requiredto effect a measurable improvement or prevention of any symptom or aparticular condition or disorder, to effect a measurable enhancement oflife expectancy, or to generally improve patient quality of life. Thetherapeutically effective amount is dependent upon the specificbiologically active molecule and the specific condition or disorder tobe treated. Therapeutically effective amounts of many proteins, such asthe mAbs described herein, are well known in the art. Thetherapeutically effective amounts of proteins not yet established or fortreating specific disorders with known proteins, such as mAbs, to beclinically applied to treat additional disorders may be determined bystandard techniques which are well within the craft of a skilledartisan, such as a physician.

The term “injectability” or “syringeability,” as generally used herein,refers to the injection performance of a pharmaceutical formulationthrough a syringe equipped with an 18-32 gauge needle, optionally thinwalled. Injectability depends upon factors such as pressure or forcerequired for injection, evenness of flow, aspiration qualities, andfreedom from clogging. Injectability of the liquid pharmaceuticalformulations may be assessed by comparing the injection force of areduced-viscosity formulation to a standard formulation without addedviscosity-lowering agents. The reduction in the injection force of theformulation containing a viscosity-lowering agent reflects improvedinjectability of that formulation. The reduced viscosity formulationshave improved injectability when the injection force is reduced by atleast 10%, preferably by at least 30%, more preferably by at least 50%,and most preferably by at least 75% when compared to a standardformulation having the same concentration of protein under otherwise thesame conditions, except for replacement of the viscosity-lowering agentwith an appropriate buffer of about the same concentration.Alternatively, injectability of the liquid pharmaceutical formulationsmay be assessed by comparing the time required to inject the samevolume, such as 0.5 mL, or more preferably about 1 mL, of differentliquid protein formulations when the syringe is depressed with the sameforce.

The term “injection force,” as generally used herein, refers to theforce required to push a given liquid formulation through a givensyringe equipped with a given needle gauge at a given injection speed.The injection force is typically reported in Newtons. For example, theinjection force may be measured as the force required to push a liquidformulation through a 1 mL plastic syringe having a 0.25 inch insidediameter, equipped with a 0.50 inch 27 gauge needle at a 250 mm/mininjection speed. Testing equipment can be used to measure the injectionforce. When measured under the same conditions, a formulation with lowerviscosity will generally require an overall lower injection force.

The “viscosity gradient,” as used herein, refers to the rate of changeof the viscosity of a protein solution as protein concentrationincreases. The viscosity gradient can be approximated from a plot of theviscosity as a function of the protein concentration for a series offormulations that are otherwise the same but have different proteinconcentrations. The viscosity increases approximately exponentially withincreasing protein concentration. The viscosity gradient at a specificprotein concentration can be approximated from the slope of a linetangent to the plot of viscosity as a function of protein concentration.The viscosity gradient can be approximated from a linear approximationto the plot of viscosity as a function of any protein concentration orover a narrow window of protein concentrations. In some embodiments aformulation is said to have a decreased viscosity gradient if, when theviscosity as a function of protein concentration is approximated as anexponential function, the exponent of the exponential function issmaller than the exponent obtained for the otherwise same formulationwithout the viscosity-lowering agent. In a similar manner, a formulationcan be said to have a lower/higher viscosity gradient when compared to asecond formulation if the exponent for the formulation is lower/higherthan the exponent for the second formulation. The viscosity gradient canbe numerically approximated from a plot of the viscosity as a functionof protein concentration by other methods known to the skilledformulation researchers.

The term “reduced-viscosity formulation,” as generally used herein,refers to a liquid formulation having a high concentration of ahigh-molecular-weight protein, such as a mAb, or a low-molecular-weightprotein that is modified by the presence of one or more additives tolower the viscosity, as compared to a corresponding formulation thatdoes not contain the viscosity-lowering additive(s).

The term “osmolarity,” as generally used herein, refers to the totalnumber of dissolved components per liter. Osmolarity is similar tomolarity but includes the total number of moles of dissolved species insolution. An osmolarity of 1 Osm/L means there is 1 mole of dissolvedcomponents per L of solution. Some solutes, such as ionic solutes thatdissociate in solution, will contribute more than 1 mole of dissolvedcomponents per mole of solute in the solution. For example, NaCldissociates into Na⁺ and Cl⁻ in solution and thus provides 2 moles ofdissolved components per 1 mole of dissolved NaCl in solution.Physiological osmolarity is typically in the range of about 280 mOsm/Lto about 310 mOsm/L.

The term “tonicity,” as generally used herein, refers to the osmoticpressure gradient resulting from the separation of two solutions by asemi-permeable membrane. In particular, tonicity is used to describe theosmotic pressure created across a cell membrane when a cell is exposedto an external solution. Solutes that can cross the cellular membrane donot contribute to the final osmotic pressure gradient. Only thosedissolved species that do not cross the cell membrane will contribute toosmotic pressure differences and thus tonicity.

The term “hypertonic,” as generally used herein, refers to a solutionwith a higher concentration of solutes than is present on the inside ofthe cell. When a cell is immersed into a hypertonic solution, thetendency is for water to flow out of the cell in order to balance theconcentration of the solutes.

The term “hypotonic,” as generally used herein, refers to a solutionwith a lower concentration of solutes than is present on the inside ofthe cell. When a cell is immersed into a hypotonic solution, water flowsinto the cell in order to balance the concentration of the solutes.

The term “isotonic,” as generally used herein, refers to a solutionwherein the osmotic pressure gradient across the cell membrane isessentially balanced. An isotonic formulation is one which hasessentially the same osmotic pressure as human blood. Isotonicformulations will generally have an osmotic pressure from about 250mOsm/kg to 350 mOsm/kg.

The term “liquid formulation,” as used herein, is a protein that iseither supplied in an acceptable pharmaceutical diluent or one that isreconstituted in an acceptable pharmaceutical diluent prior toadministration to the patient.

The terms “branded” and “reference,” when used to refer to a protein orbiologic, are used interchangeably herein to mean the single biologicalproduct licensed under section 351(a) of the U.S. Public Health ServiceAct (42 U.S.C. § 262).

The term “biosimilar,” as used herein, is generally used interchangeablywith “a generic equivalent” or “follow-on.” For example, a “biosimilarmAb” refers to a subsequent version of an innovator's mAb typically madeby a different company. “Biosimilar” when used in reference to a brandedprotein or branded biologic can refer to a biological product evaluatedagainst the branded protein or branded biologic and licensed undersection 351(k) of the U.S. Public Health Service Act (42 U.S.C. § 262).A biosimilar mAb can be one that satisfies one or more guidelinesadopted May 30, 2012 by the Committee for Medicinal Products for HumanUse (CHMP) of the European Medicines Agency and published by theEuropean Union as “Guideline on similar biological medicinal productscontaining monoclonal antibodies—non-clinical and clinical issues”(Document Reference EMA/CHMP/BMWP/403543/2010).

Biosimilars can be produced by microbial cells (prokaryotic,eukaryotic), cell lines of human or animal origin (e.g., mammalian,avian, insect), or tissues derived from animals or plants. Theexpression construct for a proposed biosimilar product will generallyencode the same primary amino acid sequence as its reference product.Minor modifications, such as N- or C-terminal truncations that will nothave an effect on safety, purity, or potency, may be present.

A biosimilar mAb is similar to the reference mAb physiochemically orbiologically both in terms of safety and efficacy. The biosimilar mAbcan be evaluated against a reference mAb using one or more in vitrostudies including assays detailing binding to target antigen(s); bindingto isoforms of the Fc gamma receptors (FcγRI, FcγRII, and FcγRIII),FcRn, and complement (C1q); Fab-associated functions (e.g.neutralization of a soluble ligand, receptor activation or blockade); orFc-associated functions (e.g. antibody-dependent cell-mediatedcytotoxicity, complement-dependent cytotoxicity, complement activation).In vitro comparisons may be combined with in vivo data demonstratingsimilarity of pharmacokinetics, pharmacodynamics, and/or safety.Clinical evaluations of a biosimilar mAb against a reference mAb caninclude comparisons of pharmacokinetic properties (e.g. AUC_(0-inf),AUC_(0-t), C_(max), t_(max), C_(trough)); pharmacodynamic endpoints; orsimilarity of clinical efficacy (e.g. using randomized, parallel groupcomparative clinical trials). The quality comparison between abiosimilar mAb and a reference mAb can be evaluated using establishedprocedures, including those described in the “Guideline on similarbiological medicinal products containing biotechnology-derived proteinsas active substance: Quality issues” (EMEA/CHMP/BWP/49348/2005), and the“Guideline on development, production, characterization andspecifications for monoclonal antibodies and related substances”

(EMEA/CHMP/BWP/157653/2007).

Differences between a biosimilar mAb and a reference mAb can includepost-translational modification, e.g. by attaching to the mAb otherbiochemical groups such as a phosphate, various lipids andcarbohydrates; by proteolytic cleavage following translation; bychanging the chemical nature of an amino acid (e.g., formylation); or bymany other mechanisms. Other post-translational modifications can be aconsequence of manufacturing process operations—for example, glycationmay occur with exposure of the product to reducing sugars. In othercases, storage conditions may be permissive for certain degradationpathways such as oxidation, deamidation, or aggregation. As all of theseproduct-related variants may be included in a biosimilar mAb.

The term “viscosity-lowering agent,” as used herein, refers to acompound which acts to reduce the viscosity of a solution relative tothe viscosity of the solution absent the viscosity-lowering agent. Theviscosity-lowering agent may be a single compound, or may be a mixtureof one or more compounds. When the viscosity-lowering agent is a mixtureof two or more compounds, the listed concentration refers to eachindividual agent, unless otherwise specified. By way of example, aformulation containing about 0.25 M camphorsulfonic acid arginine as theviscosity-lowering agent is a solution having camphorsulfonic acid at aconcentration of 0.25 M, and arginine at a concentration of 0.25 M.

Certain viscosity-lowering agents contain acidic or basic functionalgroups. Whether or not these functional groups are fully or partiallyionized depends on the pH of the formulation they are in. Unlessotherwise specified, reference to a formulation containing aviscosity-lowering agent having an ionizable functional group includesboth the parent compound and any possible ionized states.

As used herein, the term “hydrogen bond donor” refers to a hydrogen atomconnected to a relatively electronegative atom, which creates a partialpositive charge on the hydrogen atom.

As used herein, the term “hydrogen bond acceptor” refers to a relativelyelectronegative atom or functional group capable of interacting with ahydrogen atom bearing a partial positive charge.

As used herein, the term “freely rotating bond” refers to any singlybonded pair of non-hydrogen atoms.

As used herein, the term “molecular polar surface area” refers to thetotal exposed polar area on the surface of the molecule of interest. Asused herein, the term “molar volume” refers to the total volume that onemole of the molecule of interest occupies in its native state (i.e.solid, liquid).

As used herein, the term “polarizability” refers to the induced dipolemoment when the molecule of interest is placed in an electric field ofunit strength.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic acids andbases, including inorganic acids and bases, and organic acids and bases.Suitable non-toxic acids include inorganic and organic acids such asacetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaricacid, p-toluenesulfonic and the like. Suitable positively chargedcounterions include sodium, potassium, lithium, calcium and magnesium.

As used herein, the term “ionic liquid” refers to a crystalline oramorphous salt, zwitterion, or mixture thereof that is a liquid at ornear temperatures where most conventional salts are solids: at less than200° C., preferably less than 100° C. or more preferably less than 80°C. Some ionic liquids have melting temperatures around room temperature,e.g. between 10° C. and 40° C., or between 15° C. and 35° C. The term“zwitterion” is used herein to describe an overall neutrally chargedmolecule which carries formal positive and negative charges on differentchemical groups in the molecule. Examples of ionic liquids are describedin Riduan et al., Chem. Soc. Rev., 42:9055-9070, 2013; Rantwijk et al.,Chem. Rev., 107:2757-2785, 2007; Earle et al., Pure Appl. Chem.,72(7):1391-1398, 2000; and Sheldon et al., Green Chem., 4:147-151, 2002.

As used herein, the term “organophosphate” refers to a compoundcontaining one or more phosphoryl groups at least one of which iscovalently connected to an organic group through a phosphoester bond.

As used herein, a “water soluble organic dye” is an organic moleculehaving a molar solubility of at least 0.001 M at 25° C. and pH 7, andthat absorbs certain wavelengths of light, preferably in thevisible-to-infrared portion of the electromagnetic spectrum, whilepossibly transmitting or reflecting other wavelengths of light.

As used herein, the term “chalcogen” refers to Group 16 elements,including oxygen, sulfur and selenium, in any oxidation state. Forinstance, unless specified otherwise, the term “chalcogen” also includesSO₂.

As used herein, term “alkyl group” refers to straight-chain,branched-chain and cyclic hydrocarbon groups. Unless specifiedotherwise, the term alkyl group embraces hydrocarbon groups containingone or more double or triple bonds. An alkyl group containing at leastone ring system is a “cycloalkyl” group. An alkyl group containing atleast one double bond is an “alkenyl group,” and an alkyl groupcontaining at least one triple bond is an “alkynyl group.”

The term as used herein, “Aryl” refers to aromatic carbon ring systems,including fused ring systems. In an “aryl” group, each of the atoms thatform the ring are carbon atoms.

The term as used herein “Heteroaryl” refers to aromatic ring systems,including fused ring systems, wherein at least one of the atoms thatforms the ring is a heteroatom.

The term as used herein “Heterocycle” refers to ring systems that,including fused ring systems, are not aromatic, wherein at least one ofthe atoms that forms the ring is a heteroatom.

The term as used herein, “heteroatom” is any non-carbon or non-hydrogenatom. Preferred heteroatoms include oxygen, sulfur, and nitrogen.Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.

II. Formulations

Biocompatible, low-viscosity protein solutions, such as those of mAbs,can be used to deliver therapeutically effective amounts of proteins involumes useful for subcutaneous (SC) and intramuscular (IM) injections,typically less than or about 2 mL for SC and less than or about 5 mL forIM, more preferably less than or about 1 mL for SC and less than orabout 3 mL for IM. The proteins can generally have any molecular weight,although in some embodiments high-molecular-weight proteins arepreferred. In other embodiments the proteins are low-molecular-weightproteins.

Formulations may have protein concentrations between about 10 mg/mL andabout 5,000 mg/mL. The formulations, including mAb formulations, mayhave a protein concentration greater than 100 mg/mL, preferably greaterthan 150 mg/mL, more preferably greater than about 175 mg/ml, even morepreferably greater than about 200 mg/mL, even more preferably greaterthan about 225 mg/mL, even more preferably greater than about 250 mg/mL,and most preferably greater than or about 300 mg/mL. In the absence of aviscosity-lowering agent, the viscosity of a protein formulationincreases exponentially as the concentration is increased. Such proteinformulations, in the absence of a viscosity-lowering agent, may haveviscosities greater than 100 cP, greater than 150 cP, greater than 200cP, greater than 300 cP, greater than 500 cP, or even greater than 1,000cP, when measured at 25° C. Such formulations are often unsuitable forSC or IM injection. The use of one or more viscosity-lowering agentspermits the preparation of formulations having a viscosity less than orabout 100 cP, preferably less than or about 75 cP, more preferably lessthan or about 50 cP, even more preferably less than or about 30 cP, evenmore preferably less than or about 20 cP, or most preferably less thanor about 10 cP, when measured at 25° C.

Although the viscosity-lowering agents may be used to lower theviscosity of concentrated protein formulations, they may be used inless-concentrated formulations as well. In some embodiments,formulations may have protein concentrations between about 10 mg/mL andabout 100 mg/mL. The formulations may have a protein concentrationgreater than about 20 mg/mL, greater than about 40 mg/mL, or greaterthan about 80 mg/mL.

For certain proteins, formulations not having a viscosity-lowering agentmay have viscosities greater than about 20 cP, greater than about 50 cP,or greater than about 80 cP. The use of one or more viscosity-loweringagents permits the preparation of formulations having a viscosity lessthan or about 80 cP, preferably less than or about 50 cP, even morepreferably less than about 20 cP, or most preferably less than or about10 cP, when measured at 25° C.

In some embodiments, the aqueous protein formulations have a viscositythat is at least about 30% less than the analogous formulation withoutthe viscosity-lowering agent(s), when measured under the sameconditions. In other embodiments, the formulations have a viscosity thatis 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or evenmore than 90% less than the analogous formulation without theviscosity-lowering agent(s). In a preferred embodiment, the formulationcontains a therapeutically effective amount of the one or morehigh-molecular-weight proteins, such as mAbs, in a volume of less thanabout 2 mL, preferably less than about 1 mL, or more preferably lessthan about 0.75 mL.

The reduced-viscosity formulations have improved injectability andrequire less injection force compared to the analogous formulationwithout the viscosity-lowering agent (e.g., in phosphate buffer) underotherwise the same conditions. In some embodiments, the force ofinjection is decreased by more than about 20%, more than about 30%, morethan about 40%, more than about 50%, or more than about 2 fold, ascompared to standard formulations without the viscosity-loweringagent(s) under otherwise the same injection conditions. In someembodiments, the formulations possess “Newtonian flow characteristics,”defined as having viscosity which is substantially independent of shearrate. The protein formulations can be readily injected through needlesof about 18-32 gauge. Preferred needle gauges for the delivery of thelow-viscosity formulations include 27, 29, and 31 gauge, optionally thinwalled.

The formulations may contain one or more additional excipients, such asbuffers, surfactants, sugars and sugar alcohols, other polyols,preservatives, antioxidants, and chelating agents. The formulations havea pH and osmolarity suitable for administration without causingsignificant adverse side effects. In some embodiments, the concentrated,low-viscosity formulations have a pH between 5 and 8, between 5.5 and7.6, between 6.0 and 7.6, between 6.8 and 7.6, or between 5.5 and 6.5.

The low-viscosity protein formulations can allow for greater flexibilityin formulation development. The low-viscosity formulations can exhibitchanges in viscosity that are less dependent upon the proteinconcentration as compared to the otherwise same formulation without theviscosity-lowering agent. The low-viscosity protein formulations canallow for increased concentrations and decreased dosage frequencies ofthe protein. In some embodiments the low-viscosity protein formulationscontain 2 or more, 3 or more, or 4 or more different proteins. Forexample, combinations of 2 or more mAbs can be provided in a singlelow-viscosity protein formulation.

Because protein (such as mAb) formulations may be administered topatients at higher protein concentrations than otherwise similar proteinformulations not containing a viscosity-lowering agent, the dosingfrequency of the protein can be reduced. For instance, proteinspreviously requiring once daily administration may be administered onceevery two days, every three days, or even less frequently when theproteins are formulated with viscosity-lowering agents. Proteins whichcurrently require multiple administrations on the same day (either atthe same time or at different times of the day) may be administered infewer injections per day. In some instances, the frequency may bereduced to a single injection once a day. By increasing the dosageadministered per injection multiple-fold the dosing frequency can bedecreased, for example from once every 2 weeks to once every 6 weeks.

In some embodiments, the liquid formulations have a physiologicalosmolarity, for example, between about 280 mOsm/L to about 310 mOsm/L.In some embodiments, the liquid formulations have an osmolarity greaterthan about 250 mOsm/L, greater than about 300 mOsm/L, greater than about350 mOsm/L, greater than about 400 mOsm/L, or greater than about 500mOsm/L.

In some embodiments, the formulations have an osmolarity of about 200mOsm/L to about 2,000 mOsm/L or about 300 mOsm/L to about 1,000 mOsm/L.In some embodiments, the liquid formulations are essentially isotonic tohuman blood. The liquid formulations can in some cases be hypertonic.

The additives, including the viscosity-lowering agents, can be includedin any amount to achieve the desired viscosity levels of the liquidformulation, as long as the amounts are not toxic or otherwise harmful,and do not substantially interfere with the chemical and/or physicalstability of the formulation. The viscosity-lowering agent(s) in someembodiments can be independently present in a concentration less thanabout 1.0 M, preferably less than about 0.50 M, less than or equal toabout 0.30 M or less than or equal to 0.15 M. Especially preferredconcentrations include about 0.15 M and about 0.30 M. For someembodiments having two or more viscosity-lowering agents, the agents arepreferably, but not necessarily, present at the same concentration.

The viscosity-lowering agents permit faster reconstitution of alyophilized dosage unit. The dosage unit is a lyophilized cake ofprotein, viscosity-lowering agent and other excipients, to which water,saline or another pharmaceutically acceptable fluid is added. In theabsence of viscosity-lowering agents, periods of 10 minutes or more areoften required in order to completely dissolve the lyophilized cake athigh protein concentration. When the lyophilized cake contains one ormore viscosity-lowering agents, the period required to completelydissolve the cake is often reduced by a factor of two, five or even ten.In certain embodiments, less than one minute is required to completelydissolve a lyophilized cake containing greater than or about 150, 200 oreven 300 mg/mL of protein.

The low-viscosity protein formulations allow for greater flexibility informulation development. The low-viscosity formulations exhibit aviscosity that changes less with increasing protein concentrations ascompared to the otherwise same formulation without theviscosity-lowering agent(s). The low-viscosity protein formulationsexhibit a decreased viscosity gradient as compared to the otherwise sameformulation without the viscosity-lowering agent.

The viscosity gradient of the protein formulation may be 2-fold less,3-fold less, or even more than 3-fold less than the viscosity gradientof the otherwise same protein formulation without the viscosity-loweringagent(s). The viscosity gradient of the protein formulation may be lessthan 2.0 cP mL/mg, less than 1.5 cP mL/mg, less than 1.0 cP mL/mg, lessthan 0.8 cP mL/mg, less than 0.6 cP mL/mg, or less than 0.2 cP mL/mg fora protein formulation having a protein concentration between 10 mg/mLand 2,000 mg/mL. By reducing the viscosity gradient of the formulation,the protein concentration can be increased to a greater degree before anexponential increase in viscosity is observed.

A. Proteins

Any protein can be formulated, including recombinant, isolated, orsynthetic proteins, glycoproteins, or lipoproteins. These may beantibodies (including antibody fragments and recombinant antibodies),enzymes, growth factors or hormones, immunomodifiers, antiinfectives,antiproliferatives, vaccines, or other therapeutic, prophylactic, ordiagnostic proteins. In certain embodiments, the protein has a molecularweight greater than about 150 kDa, greater than 160 kDa, greater than170 kDa, greater than 180 kDa, greater than 190 kDa or even greater than200 kDa.

In certain embodiments, the protein can be a PEGylated protein. The term“PEGylated protein,” as used herein, refers to a protein having one ormore poly(ethylene glycol) or other stealth polymer groups covalentlyattached thereto, optionally through a chemical linker that may bedifferent from the one or more polymer groups. PEGylated proteins arecharacterized by their typically reduced renal filtration, decreaseduptake by the reticuloendothelial system, and diminished enzymaticdegradation leading to, for example, prolonged half-lives and enhancedbioavailability. Stealth polymers include poly(ethylene glycol);poly(propylene glycol); poly(amino acid) polymers such as poly(glutamicacid), poly(hydroxyethyl-L-asparagine), andpoly(hydroxethyl-L-glutamine); poly(glycerol); poly(2-oxazoline)polymers such as poly(2-methyl-2-oxazoline) andpoly(2-ethyl-2-oxazoline); poly(acrylamide); poly(vinylpyrrolidone);poly(N-(2-hydroxypropyl)methacrylamide); and copolymers and mixturesthereof. In preferred embodiments the stealth polymer in a PEGylatedprotein is poly(ethylene glycol) or a copolymer thereof. PEGylatedproteins can be randomly PEGylated, i.e. having one or more stealthpolymers covalently attached at non-specific site(s) on the protein, orcan be PEGylated in a site-specific manner by covalently attaching thestealth polymer to specific site(s) on the protein. Site-specificPEGylation can be accomplished, for example, using activated stealthpolymers having one or more reactive functional groups. Examples aredescribed, for instance, in Hoffman et al., Progress in Polymer Science,32:922-932, 2007.

In the preferred embodiment, the protein is high-molecular-weight and anantibody, most preferably a mAb, and has a high viscosity in aqueousbuffered solution when concentrated sufficiently to inject atherapeutically effective amount in a volume not exceeding 1.0 to 2.0 mLfor SC and 3.0 to 5.0 mL for IM administration. High-molecular-weightproteins can include those described in Scolnik, mAbs 1:179-184, 2009;Beck, mAbs 3:107-110, 2011; Baumann, Curr. Drug Meth. 7:15-21, 2006; orFederici, Biologicals 41:131-147, 2013. The proteins for use in theformulations described herein are preferably essentially pure andessentially homogeneous (i.e., substantially free from contaminatingproteins and/or irreversible aggregates thereof).

Preferred mAbs herein include natalizumab (TYSABRI®), cetuximab(ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab(REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab(ARZERRA®), and biosimilars thereof. Exemplary high-molecular-weightproteins can include tocilizumab (ACTEMRA®), alemtuzumab (marketed underseveral trade names), brodalumab (developed by Amgen, Inc (“Amgen”)),denosumab (PROLIA® and XGEVA®), and biosimilars thereof.

Exemplary molecular targets for antibodies described herein include CDproteins, such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HERreceptor family such as the EGF receptor, HER2, HER3 or HER4 receptor;cell adhesion molecules, such as LFA-1, Mol, p150,95, VLA-4, ICAM-1,VCAM, and αv/β3 integrin, including either α or β subunits thereof(e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factors,such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity(OB) receptor; protein C; PCSK9; etc.

Antibody Therapeutics Currently on the Market

Many protein therapeutics currently on the market, especially antibodiesas defined herein, are administered via IV infusions due to high dosingrequirements. Formulations can include one of the antibody therapeuticscurrently on the market or a biosimilar thereof. Some proteintherapeutics currently on the market are not high-molecular-weight, butare still administered via IV infusion because high doses are needed fortherapeutic efficacy. In some embodiments, liquid formulations areprovided of these low-molecular-weight proteins as defined herein withconcentrations to deliver therapeutically effective amounts for SC or IMinjections.

Antibody therapeutics currently on the market include belimumab(BENLYSTA®), golimumab (SIMPONI ARIA®), abciximab (REOPRO®), thecombination of tositumomab and iodine-131 tositumomab, marketed asBEXXAR®, alemtuzumab (CAMPATH®), palivizumab (SYNAGIS®), basiliximab(SIMULECT®), ado-trastuzumab emtansine (KADCYLA®), pertuzumab(PERJETA®), capromab pendetide (PROSTASCINT KIT®), caclizumab(ZENAPAX®), ibritumomab tiuxetan)(ZEVALIN®, eculizumab (SOLIRIS®),ipilimumab (YERVOY®), muromonab-CD3 (ORTHOCLONE OKT3®), raxibacumab,nimotuzumab (THERACIM®), brentuximab vedotin (ADCETRIS®), adalimumab(HUMIRA®), golimumab (SIMPONI®), palivizumab (SYNAGIS®), omalizumab(XOLAIR®), and ustekinumab (STELARA®).

Natalizumab, a humanized mAb against the cell adhesion moleculeα4-integrin, is used in the treatment of multiple sclerosis and Crohn'sdisease. Previously marketed under the trade name ANTEGREN®, natalizumabis currently co-marketed as TYSABRI® by Biogen Idec (“Biogen”) and ElanCorp. (“Elan”) TYSABRI® is produced in murine myeloma cells. Each 15 mLdose contains 300 mg natalizumab; 123 mg sodium chloride, USP; 17.0 mgsodium phosphate, monobasic, monohydrate, USP; 7.24 mg sodium phosphate,dibasic, heptahydrate, USP; 3.0 mg polysorbate 80, USP/NF, in water forIV injection, USP at pH 6.1. Natalizumab is typically administered bymonthly intravenous (IV) infusions and has been proven effective intreating the symptoms of both multiple sclerosis and Crohn's disease, aswell as for preventing relapse, vision loss, cognitive decline, andsignificantly improving patient's quality of life.

As used herein, the term “natalizumab” includes the mAb against the celladhesion molecule α4-integrin known under the InternationalNonproprietary Name “NATALIZUMAB” or an antigen binding portion thereof.Natalizumab includes antibodies described in U.S. Pat. No. 5,840,299,U.S. Pat. No. 6,033,665, U.S. Pat. No. 6,602,503, U.S. Pat. No.5,168,062, U.S. Pat. No. 5,385,839, and U.S. Pat. No. 5,730,978.Natalizumab includes the active agent in products marketed under thetrade name TYSABRI® by Biogen Idec and Elan Corporation or a biosimilarproduct thereof.

Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor usedfor the treatment of metastatic colorectal cancer and head and neckcancer. Cetuximab is a chimeric (mouse/human) mAb typically given by IVinfusion. Cetuximab is marketed for IV use only under the trade nameERBITUX® by Bristol-Myers Squibb Company (North America; “Bristol-MyersSquibb”), Eli Lilly and Company (North America; “Eli Lilly”), and MerckKGaA. ERBITUX® is produced in mammalian (murine myeloma) cell culture.Each single-use, 50-mL vial of ERBITUX® contains 100 mg of cetuximab ata concentration of 2 mg/mL and is formulated in a preservative-freesolution containing 8.48 mg/mL sodium chloride, 1.88 mg/mL sodiumphosphate dibasic heptahydrate, 0.42 mg/mL sodium phosphate monobasicmonohydrate, and water for IV Injection, USP.

Cetuximab is indicated for the treatment of patients with epidermalgrowth factor receptor (EGFR)-expressing, KRAS wild-type metastaticcolorectal cancer (mCRC), in combination with chemotherapy, and as asingle agent in patients who have failed oxaliplatin- andirinotecan-based therapy or who are intolerant to irinotecan. Cetuximabis indicated for the treatment of patients with squamous cell carcinomaof the head and neck in combination with platinum-based chemotherapy forthe first-line treatment of recurrent and/or metastatic disease and incombination with radiation therapy for locally advanced disease.Approximately 75% of patients with metastatic colorectal cancer have anEGFR-expressing tumor and are, therefore, considered eligible fortreatment with cetuximab or panitumumab, according to FDA guidelines.

As used herein, the term “cetuximab” includes the mAb known under theInternational Nonproprietary Name “CETUXIMAB” or an antigen bindingportion thereof. Cetuximab includes antibodies described in U.S. Pat.No. 6,217,866. Cetuximab includes the active agent in products marketedunder the trade name ERBITUX® and biosimilar products thereof.Biosimilars of ERBITUX® can include those currently being developed byAmgen, AlphaMab Co., Ltd. (“AlphaMab”), and Actavis plc (“Actavis”).

Bevacizumab, a humanized mAb that inhibits vascular endothelial growthfactor A (VEGF-A), acts as an anti-angiogenic agent. It is marketedunder the trade name AVASTIN® by Genentech, Inc. (“Genentech”) and F.Hoffmann-La Roche, LTD (“Roche”). It is licensed to treat variouscancers, including colorectal, lung, breast (outside the U.S.A.),glioblastoma (U.S.A. only), kidney and ovarian. AVASTIN® was approved bythe FDA in 2004 for use in metastatic colorectal cancer when used withstandard chemotherapy treatment (as first-line treatment) and with5-fluorouracil-based therapy for second-line metastatic colorectalcancer. In 2006, the FDA approved AVASTIN® for use in first-lineadvanced non-squamous non-small cell lung cancer in combination withcarboplatin/paclitaxel chemotherapy. AVASTIN® is given as an IV infusionevery three weeks at the dose of either 15 mg/kg or 7.5 mg/kg. Thehigher dose is usually given with carboplatin-based chemotherapy,whereas the lower dose is given with cisplatin-based chemotherapy. In2009, the FDA approved AVASTIN® for use in metastatic renal cellcarcinoma (a form of kidney cancer). The FDA also granted acceleratedapproval of AVASTIN® for the treatment of recurrent glioblastomamultiforme in 2009. Treatment for initial growth is still in phase IIIclinical trial.

The National Comprehensive Cancer Network (“NCCN”) recommendsbevacizumab as standard first-line treatment in combination with anyplatinum-based chemotherapy, followed by maintenance bevacizumab untildisease progression. The NCCN updated its Clinical Practice Guidelinesfor Oncology (NCCN Guidelines) for Breast Cancer in 2010 to affirm therecommendation regarding the use of bevacizumab (AVASTIN®,Genentech/Roche) in the treatment of metastatic breast cancer.

As used herein, the term “bevacizumab” includes the mAb that inhibitsvascular endothelial growth factor A (VEGF-A) known under theInternational Nonproprietary Name/Common Name “BEVACIZUMAB” or anantigen binding portion thereof. Bevacizumab is described in U.S. Pat.No. 6,054,297. Bevacizumab includes the active agent in productsmarketed under the trade name AVASTIN® and biosimilar products thereof.Biosimilars of AVASTIN® can include those currently being developed byAmgen, Actavis, AlphaMab, and Pfizer, Inc (“Pfizer”). Biosimilars ofAVASTIN® can include the biosimilar known as BCD-021 produced by Biocadand currently in clinical trials in the U.S.

Trastuzumab is a mAb that interferes with the HER2/neu receptor.Trastuzumab is marketed under the trade name HERCEPTIN® by Genentech,Inc. HERCEPTIN® is produced by a mammalian cell (Chinese Hamster Ovary(CHO)) line. HERCEPTIN® is a sterile, white to pale-yellow,preservative-free lyophilized powder for IV administration. EachHERCEPTIN® vial contains 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4mg L-histidine, 400 mg a,a-trehalose dihydrate, and 1.8 mg polysorbate20, USP. Reconstitution with 20 mL water yields a multi-dose solutioncontaining 21 mg/mL trastuzumab. HERCEPTIN® is currently administeredvia IV infusion as often as weekly and at a dosage ranging from about 2mg/kg to about 8 mg/kg.

Trastuzumab is mainly used to treat certain breast cancers. The HER2gene is amplified in 20-30% of early-stage breast cancers, which makesit overexpress epidermal growth factor (EGF) receptors in the cellmembrane. Trastuzumab is generally administered as a maintenance therapyfor patients with HER2-positive breast cancer, typically for one yearpost-chemotherapy. Trastuzumab is currently administered via IV infusionas often as weekly and at a dosage ranging from about 2 mg/kg to about 8mg/kg.

As used herein, the term “trastuzumab” includes the mAb that interfereswith the HER2/neu receptor known under the International NonproprietaryName/Common Name “TRASTUZUMAB” or an antigen binding portion thereof.Trastuzumab is described in U.S. Pat. No. 5,821,337. Trastuzumabincludes the active agent in products marketed under the trade nameHERCEPTIN® and biosimilars thereof. The term “trastuzumab” includes theactive agent in biosimilar HERCEPTIN® products marketed under the tradenames HERTRAZ® by Mylan, Inc. (“Mylan”) and CANMAB® by Biocon, Ltd.(“Biocon”). Trastuzumab can include the active agent in biosimilarHERCEPTIN® products being developed by Amgen and by PlantFormCorporation, Canada.

Infliximab is a mAb against tumor necrosis factor alpha (TNF-α) used totreat autoimmune diseases. It is marketed under the trade name REMICADE®by Janssen Global Services, LLC (“Janssen”) in the U.S., MitsubishiTanabe Pharma in Japan, Xian Janssen in China, and Merck & Co (“Merck”);elsewhere. Infliximab is a chimeric mouse/human monoclonal antibody witha high molecular weight of approximately 144 kDa. In some embodiments,the formulations contain a biosimilar of REMICADE®, such as REMSIMA™ orINFLECTRA™. Both REMSIMA™, developed by Celltrion, Inc. (“Celltrion”),and INFLECTRA™, developed by Hospira Inc., UK, have been recommended forregulatory approval in Europe. Celltrion has submitted a filing forREMSIMA™ to the FDA. Infliximab is currently administered via IVinfusion at doses ranging from about 3 mg/kg to about 10 mg/kg.

Infliximab contains approximately 30% murine variable region amino acidsequence, which confers antigen-binding specificity to human TNFα. Theremaining 70% correspond to a human IgG1 heavy chain constant region anda human kappa light chain constant region. Infliximab has high affinityfor human TNFα, which is a cytokine with multiple biologic actionsincluding mediation of inflammatory responses and modulation of theimmune system.

Infliximab is a recombinant antibody generally produced and secretedfrom mouse myeloma cells (SP2/0 cells). The antibody is currentlymanufactured by continuous perfusion cell culture. The infliximabmonoclonal antibody is expressed using chimeric antibody genesconsisting of the variable region sequences cloned from the murineanti-TNFα hybridoma A2, and human antibody constant region sequencessupplied by the plasmid expression vectors. Generation of the murineanti-TNF α hybridoma is performed by immunization of BALB/c mice withpurified recombinant human TNFα. The heavy and light chain vectorconstructs are linearized and transfected into the Sp2/0 cells byelectroporation. Standard purification steps can include chromatographicpurification, viral inactivation, nanofiltration, andultrafiltration/diafiltration.

As used herein, the term “infliximab” includes the chimeric mouse/humanmonoclonal antibody known under the International Nonproprietary Name“INFLIXIMAB” or an antigen binding portion thereof. Infliximabneutralizes the biological activity of TNFα by binding with highaffinity to the soluble and transmembrane forms of TNFα and inhibitsbinding of TNFα with its receptors. Infliximab is described in U.S. Pat.No. 5,698,195. The term “Infliximab” includes the active agent inproducts marketed or proposed to be marketed under the trade namesREMICADE® by multiple entities; REMSIMA™ by Celltrion and INFLECTRA™ byHospira, Inc (“Hospira”). Infliximab is supplied as a sterilelyophilized cake for reconstitution and dilution. Each vial ofinfliximab contains 100 mg infliximab and excipients such as monobasicsodium phosphate monohydrate, dibasic sodium phosphate dihydrate,sucrose, and polysorbate 80.

Denosumab (PROLIA® and XGEVA®) is a human mAb—and the first RANKLinhibitor—approved for use in postmenopausal women with risk ofosteoporosis and patients with bone metastases from solid tumors.Denosumab is in Phase II trials for the treatment of rheumatoidarthritis.

Panitumumab is a fully human mAb approved by the FDA for treatment ofEGFR-expressing metastatic cancer with disease progression. Panitumumabis marketed under the trade name VECTIBIX® by Amgen. VECTIBIX® ispackaged as a 20 mg/ml panitumumab concentrate in 5 ml, 10 ml, and 15 mlvials for IV infusion. When prepared according to the packaginginstructions, the final panitumumab concentration does not exceed 10mg/ml. VECTIBIX® is administered at a dosage of 6 mg/kg every 14 days asan intravenous infusion. As used herein, the term “panitumumab” includesthe anti-human epidermal growth factor receptor known by theInternational Nonproprietary Name “PANITUMUMAB.” The term “panitumumab”includes the active agent in products marketed under the trade nameVECTIBIX® by Amgen and biosimilars thereof. The term “panitumumab”includes monoclonal antibodies described in U.S. Pat. No. 6,235,883. Theterm “panitumumab” includes the active agent in biosimilar VECTIBIX®products, including biosimilar VECTIBIX® being developed by BioXpress,SA (“BioXpress”).

Belimumab (BENLYSTA®) is a human mAb with a molecular weight of about151.8 kDa that inhibits B-cell activating factor (BAFF). Belimumab isapproved in the United States, Canada, and Europe for treatment ofsystemic lupus erythematosus. Belimumab is currently administered tolupus patients by IV infusion at a 10 mg/kg dosage. Ahigh-molecular-weight, low-viscosity protein formulation can includeBelimumab, preferably in a concentration of about 400 mg/mL to about1,000 mg/mL. The preferred ranges are calculated based upon body weightof 40-100 kg (approximately 80-220 lbs) in a 1 mL volume.

Abciximab (REOPRO®) is manufactured by Janssen Biologics BV anddistributed by Eli Lilly & Company (“Eli Lilly”). Abciximab is a Fabfragment of the chimeric human-murine monoclonal antibody 7E3. Abciximabbinds to the glycoprotein (GP) IIb/IIIa receptor of human platelets andinhibits platelet aggregation by preventing the binding of fibrinogen,von Willebrand factor, and other adhesive molecules. It also binds tovitronectin (αvβ3) receptor found on platelets and vessel wallendothelial and smooth muscle cells. Abciximab is a platelet aggregationinhibitor mainly used during and after coronary artery procedures.Abciximab is administered via IV infusion, first in a bolus of 0.25mg/kg and followed by continuous IV infusion of 0.125 mcg/kg/minute for12 hours.

Tositumomab (BEXXAR®) is a drug for the treatment of follicularlymphoma. It is an IgG2a anti-CD20 mAb derived from immortalized mousecells. Tositumomab is administered in sequential infusions: cold mAbfollowed by iodine (131I) tositumomab, the same antibody covalentlybound to the radionuclide iodine-131. Clinical trials have establishedthe efficacy of the tositumomab/iodine tositumomab regimen in patientswith relapsed refractory follicular lymphoma. BEXXAR® is currentlyadministered at a dose of 450 mg via IV infusion.

Alemtuzumab (marketed as CAMPATH®, MABCAMPATH®, or CAMPATH-1H® andcurrently under further development as LEMTRADA®) is a mAb used in thetreatment of chronic lymphocytic leukemia (CLL), cutaneous T-celllymphoma (CTCL), and T-cell lymphoma. It is also used under clinicaltrial protocols for treatment of some autoimmune diseases, such asmultiple sclerosis. Alemtuzumab has a weight of approximately 145.5 kDa.It is administered in daily IV infusions of 30 mg for patients withB-cell chronic lymphocytic leukemia.

Palivizumab (SYNAGIS®) is a humanized mAb directed against an epitope inthe A antigenic site of the F protein of respiratory syncytial virus. Intwo Phase III clinical trials in the pediatric population, palivizumabreduced the risk of hospitalization due to respiratory syncytial virusinfection by 55% and 45%. Palivizumab is dosed once a month via IMinjection of 15 mg/kg.

Ofatumumab is a human anti-CD20 mAb which appears to inhibit early-stageB lymphocyte activation. Ofatumumab is marketed under the trade nameARZERRA® by GlaxoSmithKline, plc (“GlaxoSmithKline”). ARZERRA® isdistributed in single-use vials containing 100 mg/5 mL and 1,000 mg/50mL ofatumumab for IV infusion. Ofatumumab is FDA-approved for treatingchronic lymphocytic leukemia and has also shown potential in treatingFollicular non-Hodgkin's lymphoma, Diffuse large B cell lymphoma,rheumatoid arthritis, and relapsing remitting multiple sclerosis.Ofatumumab has a molecular weight of about 149 kDa. It is currentlyadministered by IV infusion at an initial dose of 300 mg, followed byweekly infusions of 2,000 mg. As used herein, the term “ofatumumab”includes the anti-CD20 mAb known by the International NonproprietaryName “OFATUMUMAB.” The term “ofatumumab” includes the active agent inproducts marketed under the trade name ARZERRA® and biosimilars thereof.The term “ofatumumab” includes the active agent in biosimilar ARZERRA®products being developed by BioExpress. High-molecular-weight,low-viscosity liquid protein formulations can include ofatumumab,preferably in a concentration of about 300 mg/mL to about 2,000 mg/mL.

Trastuzumab emtansine (in the U.S., ado-trastuzumab emtansine, marketedas KADCYLA®) is an antibody-drug conjugate consisting of the mAbtrastuzumab linked to the cytotoxic agent mertansine (DM1®).Trastuzumab, described above, stops growth of cancer cells by binding tothe HER2/neu receptor, whereas mertansine enters cells and destroys themby binding to tubulin. In the United States, trastuzumab emtansine wasapproved specifically for treatment of recurring HER2-positivemetastatic breast cancer. Multiple Phase III trials of trastuzumabemtansine are planned or ongoing in 2014. Trastuzumab emtansine iscurrently administered by IV infusion of 3.6 mg/kg.High-molecular-weight, low-viscosity liquid formulations can includetrastuzumab emtansine, preferably in a concentration of about 144 mg/mLto about 360 mg/mL.

Pertuzumab (PERJETA®) is a mAb that inhibits HER2 dimerization.Pertuzumab received FDA approval for the treatment of HER2-positivemetastatic breast cancer in 2012. The currently recommended dosage ofPertuzumab is 420 mg to 840 mg by IV infusion. High-molecular-weight,low-viscosity liquid formulations can include pertuzumab, preferably ina concentration of about 420 mg/mL to about 840 mg/mL.

Daclizumab is a humanized anti-CD25 mAb and is used to prevent rejectionin organ transplantation, especially in kidney transplants. The drug isalso under investigation for the treatment of multiple sclerosis.Daclizumab has a molecular weight of about 143 kDa. Daclizumab wasmarketed in the U.S. by Hoffmann-La Roche, Ltd. (“Roche”) as ZENAPAX®and administered by IV infusion of 1 mg/kg. Daclizumab High-YieldProcess (DAC HYP; BIIB019; Biogen Idec (“Biogen”) and AbbVie, Inc.(“AbbVie”)) is in phase III clinical trials as a 150 mg, once-monthlysubcutaneous injection to treat relapsing, remitting multiple-sclerosis.High-molecular-weight, low-viscosity liquid formulations can includedaclizumab, preferably in a concentration of about 40 mg/mL to about 300mg/mL.

Eculizumab (SOLIRIS®) is a humanized mAb approved for the treatment ofrare blood diseases, such as paroxysmal nocturnal hemoglobinuria andatypical hemolytic uremic syndrome. Eculizumab, with a molecular weightof about 148 kDa, is being developed by Alexion Pharmaceuticals, Inc(“Alexion”). It is administered by IV infusion in the amount of about600 mg to about 1,200 mg. High-molecular-weight, low-viscosity liquidformulations can include eculizumab, preferably in a concentration ofabout 500 mg/mL to about 1,200 mg/mL.

Tocilizumab (ACTEMRA®) is a humanized mAb against the interleukin-6receptor. It is an immunosuppressive drug, mainly for the treatment ofrheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, asevere form of RA in children. Tocilizumab is commonly administered byIV infusion in doses of about 6 mg/kg to about 8 mg/kg.High-molecular-weight, low-viscosity liquid formulations can includetocilizumab, preferably in a concentration of about 240 mg/mL to about800 mg/mL.

Rituximab (RITUXAN®) is a chimeric anti-CD20 mAb used to treat a varietyof diseases characterized by excessive numbers of B cells, overactive Bcells, or dysfunctional B cells. Rituximab is used to treat cancers ofthe white blood system, such as leukemias and lymphomas, includingHodgkin's lymphoma and its lymphocyte-predominant subtype. It has beenshown to be an effective rheumatoid arthritis treatment. Rituximab iswidely used off-label to treat difficult cases of multiple sclerosis,systemic lupus erythematosus, and autoimmune anemias.

Rituximab is jointly marketed in the U.S. under the trade name RITUXAN®by Biogen and Genentech and outside the U.S. under the trade nameMABTHERA® by Roche. RITUXAN® is distributed in single-use vialscontaining 100 mg/10 mL and 500 mg/50 mL. RITUXAN® is typicallyadministered by IV infusion of about 375 mg/m². The term “rituximab,” asused herein, includes the anti-CD20 mAb known under the InternationalNonproprietary Name/Common Name “RITUXIMAB.” Rituximab includes mAbsdescribed in U.S. Pat. No. 5,736,137. Rituximab includes the activeagent in products marketed under the trade name RITUXAN® and MABTHERA®and biosimilars thereof.

High-molecular-weight, low-viscosity liquid formulations can includerituximab, preferably in a concentration of about 475 mg/mL to about 875mg/mL (approximated using a body surface area range of 1.3 to 2.3 squaremeters, derived from the Mosteller formula for persons ranging from 5ft, 40 kg to 6 ft, 100 kg). Concentrations are calculated for a 1 mLformulation.

Ipilimumab is a human mAb developed by Bristol-Myers Squibb Company(“Bristol-Myers Squibb”). Marketed as YERVOY®, it is used for thetreatment of melanoma and is also undergoing clinical trials for thetreatment of non-small cell lung carcinoma (NSCLC), small cell lungcancer (SCLC), and metastatic hormone-refractory prostate cancer.Ipilimumab is currently administered by IV infusion of 3 mg/kg.High-molecular-weight, low-viscosity liquid formulations can includeipilimumab, preferably in a concentration of about 120 mg/mL to about300 mg/mL.

Raxibacumab (ABthrax®) is a human mAb intended for the prophylaxis andtreatment of inhaled anthrax. It is currently administered by IVinfusion. The suggested dosage in adults and children over 50 kg is 40mg/kg. High-molecular-weight, low-viscosity liquid formulations caninclude raxibacumab, preferably in a concentration of about 1,000 mg/mLto about 4,000 mg/mL.

Nimotuzumab (THERACIM®, BIOMAB EGFR®, THERALOC®, CIMAher®) is ahumanized mAb with a molecular weight of about 151 kDa used to treatsquamous cell carcinomas of the head and neck, recurrent or refractoryhigh-grade malignant glioma, anaplastic astrocytomas, glioblastomas, anddiffuse intrinsic pontine glioma. Nimotuzumab is typically administeredby IV infusion of about 200 mg weekly. High-molecular-weight,low-viscosity liquid formulations can include nimotuzumab, preferably ina concentration of about 200 mg/mL.

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate directedto the protein CD30, expressed in classical Hodgkin's lymphoma andsystemic anaplastic large cell lymphoma. It is administered by IVinfusion of about 1.8 mg/kg. High-molecular-weight, low-viscosity liquidformulations can include brentuximab vedotin, preferably in aconcentration of about 80 mg/mL to about 200 mg/mL.

Itolizumab (ALZUMAB®) is a humanized IgG1 mAb developed by Biocon.Itolizumab completed successful Phase III studies in patients withmoderate to severe psoriasis. Itolizumab has received marketing approvalin India; an application for FDA approval has not been submitted.

Obinutuzumab (GAZYVA®), originally developed by Roche and being furtherdeveloped under a collaboration agreement with Biogen is a humanizedanti-CD20 mAb approved for treatment of chronic lymphocytic leukemia. Itis also being investigated in Phase III clinical trials for patientswith various lymphomas. Dosages of about 1,000 mg are being administeredvia IV infusion.

Certolizumab pegol (CIMZIA®) is a recombinant, humanized antibody Fab′fragment, with specificity for human tumor necrosis factor alpha (TNFα),conjugated to an approximately 40 kDa polyethylene glycol (PEG2MAL40K).The molecular weight of certolizumab pegol is approximately 91 kDa.

Other antibody therapeutics that can be formulated withviscosity-lowering agents include CT-P6 from Celltrion, Inc.(Celltrion).

Antibody Therapeutics in Late-Stage Trials and Development

The progression of antibody therapeutics to late-stage clinicaldevelopment and regulatory review are proceeding at a rapid pace. In2014, there are more than 300 mAbs in clinical trials and 30commercially-sponsored antibody therapeutics undergoing evaluation inlate-stage studies. First marketing applications for two mAbs(vedolizumab and ramucirumab) were recently submitted to the FDA. Amgenis currently sponsoring multiple ongoing Phase III trials on the use ofbrodalumab in patients with plaque psoriasis, with additional trialsplanned or recruiting patients. XBiotech, Inc. has sponsored two Phase Iclinical trials of MABp1 (Xilonix) for patients with advanced cancer ortype-2 diabetes. Additional trials of MABp1 are recruiting patients.Multiple trials are sponsored by MedImmune, LLC (“MedImmune”) andunderway or recruiting patients for the treatment of leukemia withmoxetumomab pasudotox. Long-term safety and efficacy studies areunderway for the use of tildrakizumab for the treatment of chronicplaque psoriasis. Multiple phase II trials have recently completed forthe use of rilotumumab for the treatment of various cancers.

At least 28 mAbs are high-molecular-weight proteins currently in orhaving recently completed Phase III studies for the treatment ofinflammatory or immunological disorders, cancers, high cholesterol,osteoporosis, Alzheimer's disease, and infectious diseases. The mAbs inor having recently completed Phase III trials include AMG 145,elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerumab(RG1450), gevokizumab, inotuzumab ozogamicin, itolizumab, ixekizumab,lebrikizumab, mepolizumab, naptumomab estafenatox, necitumumab,nivolumab, ocrelizumab, onartuzumab, racotumomab, ramucirumab,reslizumab, romosozumab, sarilumab, secukinumab, sirukumab, solanezumab,tabalumab, and vedolizumab. A mAb mixture (actoxumab and bezlotoxumab)is also being evaluated in Phase III trials. See, e.g., Reichert, MAbs5:1-4, 2013.

Vedolizumab is a mAb being developed by Millennium Pharmaceuticals, Inc(“Millennium”; a subsidiary of Takeda Pharmaceuticals Company, Ltd.(“Takeda”)). Vedolizumab was found safe and highly effective forinducing and maintaining clinical remission in patients with moderate tosevere ulcerative colitis. Phase III clinical trials showed it to meetthe objectives of inducing a clinical response and maintaining remissionin Crohn's and ulcerative colitis patients. Studies evaluating long-termclinical outcomes show close to 60% of patients achieving clinicalremission. A common dose of vedolizumab are 6 mg/kg by IV infusion.

Ramucirumab is a human mAb being developed for the treatment of solidtumors. Phase III clinical trials are ongoing for the treatment ofbreast cancer, metastatic gastric adenocarcinoma, non-small cell lungcancer, and other types of cancer. Ramucirumab, in some Phase IIItrials, is administered at about 8 mg/kg via IV infusion.

Rilotumumab is a human mAb that inhibits the action of hepatocyte growthfactor/scatter factor. Developed by Amgen, it is in Phase III trials asa treatment for solid tumors. An open Phase III study of rilotumumabtreatment in patients with advanced or metastatic esophageal cancer willadminister rilotumumab at about 15 mg/kg via IV infusion.

Evolocumab (AMG 145), also developed by Amgen, is a mAb that binds toPCSK9. Evolocumab is indicated for hypercholesterolemia andhyperlipidemia.

Alirocumab (REGN727) is a human mAb from Regeneron Pharmaceuticals, Inc.(“Regeneron”) and Sanofi-Aventis U.S. LLC (“Sanofi”), indicated forhypercholesterolemia and acute coronary syndrome.

Naptumomab estafenatox, ABR-217620 from Active Biotech AB (“ActiveBiotech”) is a mAb indicated for renal cell carcinoma.

Racotumomab from CIMAB, SA (“CIMAB”); Laboratorio Elea S.A.C.I.F.y A. isa mAb indicated for non-small cell lung cancer.

Other antibodies which may be formulated with viscosity-lowering agentsinclude bococizumab (PF-04950615) and tanezumab; ganitumab,blinatumomab, trebananib from Amgen; Anthrax immune globulin fromCangene Corporation; teplizumab from MacroGenics, Inc.; MK-3222, MK-6072from Merck & Co (“Merck”); girentuximab from Wilex AG; RIGScan fromNavidea Biopharmaceuticals (“Navidea”); PF-05280014 from Pfizer; SA237from Chugai Pharmaceutical Co. Ltd. (“Chugai”); guselkumab fromJanssen/Johnson and Johnson Services, Inc. (“J&J”); Antithrombin Gamma(KW-3357) from Kyowa; and CT-P10 from Celltrion.

Antibodies in Early-Stage Clinical Trials

Many mAbs have recently entered, or are entering, clinical trials. Theycan include proteins currently administered via IV infusion, preferablythose having a molecular weight greater than about 120 kDa, typicallyfrom about 140 kDa to about 180 kDa. They can also include suchhigh-molecular-weight proteins such as Albumin-conjugated drugs orpeptides that are also entering clinical trials or have been approved bythe FDA. Many mAbs from Amgen are currently in clinical trials. Thesecan be high-molecular-weight proteins, for example, AMG 557, which is ahuman monoclonal antibody developed jointly by Amgen and AstraZeneca andcurrently in Phase I trials for treatment of lupus. Likewise, AMG 729 isa humanized mAb developed by Amgen and currently in Phase I trials forthe treatment of lupus and rheumatoid arthritis. In addition, AMG 110 isa mAb for epithelial cell adhesion molecule; AMG 157, jointly developedby Amgen and AstraZeneca, is a human mAb currently in Phase I for thetreatment of asthma; AMG 167 is a humanized mAb that has been evaluatedin multiple Phase I trials for the treatment of osteopenia; AMG 334,having completed Phase I dosing studies and currently in in Phase IIstudies for the treatment of migraines and hot flashes, is a human mAbthat inhibits Calcitonin Gene-Related Peptide; AMG 780 is a humananti-angiopoietin mAb that inhibits the interaction between theendothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2,and recently completed Phase I trials as a cancer treatment; AMG 811 isa human monoclonal antibody that inhibits interferon gamma beinginvestigated as a treatment for systemic lupus erythematosus; AMG 820 isa human mAb that inhibits c-fms and decreases tumor associatedmacrophage (TAM) function and is being investigated as a cancertreatment; AMG 181, jointly developed by Amgen and AstraZeneca, is ahuman mAb that inhibits the action of alpha4/beta7 and is in Phase IItrials as a treatment for ulcerative colitis and Crohn's disease.

Many mAbs are currently in clinical trials for the treatment ofautoimmune disorders. These mAbs can be included in low-viscosity,high-molecular-weight liquid formulations. RG7624 is a fully human mAbdesigned to specifically and selectively bind to the humaninterleukin-17 family of cytokines A Phase I clinical trial evaluatingRG7624 for autoimmune disease is ongoing. BIIB033 is an anti-LINGO-1 mAbby Biogen currently in Phase II trials for treating multiple sclerosis.

High-molecular-weight proteins also can include AGS-009, a mAb targetingIFN-alpha developed by Argos Therapeutics, Inc. that recently completedphase I trials for the treatment of lupus. Patients are administered upto 30 mg/kg of AGS-009 via IV infusion. BT-061, developed by AbbVie, isin Phase II trials for patients with rheumatoid arthritis. Certolizumabpegol (CIMZIA®) is a mAb in Phase II trials for ankylosing spondylitisand juvenile rheumatoid arthritis. Clazakizumab, an anti-IL6 mAb, is inPhase II trials by Bristol-Myers Squibb.

CNTO-136 (sirukumab) and CNTO-1959 are mABs having recently completedPhase II and Phase III trials by Janssen. Daclizumab (previouslymarketed as ZENAPAX® by Roche) is currently in or has recently completedmultiple Phase III trials by AbbVie for the treatment of multiplesclerosis. Epratuzumab is a humanized mAb in Phase III trials for thetreatment of lupus. Canakinumab (ILARIS®) is a human mAb targeted atinterleukin-1 beta. It was approved for the treatment ofcryopyrin-associated periodic syndromes. Canakinumab is in Phase Itrials as a possible treatment for chronic obstructive pulmonarydisease, gout and coronary artery disease. Mavrilimumab is a human mAbdesigned for the treatment of rheumatoid arthritis. Discovered asCAM-3001 by Cambridge Antibody Technology, mavrilimumab is beingdeveloped by MedImmune.

MEDI-546 are MEDI-570 are mAbs currently in Phase I and Phase II trialsby AstraZeneca for the treatment of lupus. MEDI-546 is administered inthe Phase II study by regular IV infusions of 300-1,000 mg. MEDI-551,another mAb being developed by AstraZeneca for numerous indications, isalso currently administered by IV infusion. NN8209, a mAb blocking theC5aR receptor being developed by Novo Nordisk A/S (“Novo Nordisk”), hascompleted a Phase II dosing study for treatment of rheumatoid arthritis.NN8210 is another antiC5aR mAb being developed by Novo Nordisk andcurrently is in Phase I trials. IPH2201 (NN8765) is a humanized mAbtargeting NKG2A being developed by Novo Nordisk to treat patients withinflammatory conditions and autoimmune diseases. NN8765 recentlycompleted Phase I trials.

Olokizumab is a humanized mAb that potently targets the cytokine IL-6.IL-6 is involved in several autoimmune and inflammatory pathways.Olokizumab has completed Phase II trials for the treatment of rheumatoidarthritis. Otelixizumab, also known as TRX4, is a mAb, which is beingdeveloped for the treatment of type 1 diabetes, rheumatoid arthritis,and other autoimmune diseases. Ozoralizumab is a humanized mAb that hascompleted Phase II trials.

Pfizer currently has Phase I trials for the mAbs PD-360324 andPF-04236921 for the treatment of lupus. A rituximab biosimilar,PF-05280586, has been developed by Pfizer and is in Phase I/Phase IItrials for rheumatoid arthritis.

Rontalizumab is a humanized mAb being developed by Genentech. Itrecently completed Phase II trials for the treatment of lupus. SAR113244(anti-CXCR5) is a mAb by Sanofi in Phase I trials. Sifalimumab(anti-IFN-alpha mAb) is a mAb in Phase II trials for the treatment oflupus.

A high-molecular-weight low-viscosity liquid formulation can include oneof the mAbs in early stage clinical development for treating variousblood disorders. For example, Belimumab (BENLYSTA®) has recentlycompleted Phase I trials for patients with vasculitis. Other mAbs inearly-stage trials for blood disorders include BI-655075 from BoehringerIngelheim GmbH “Boehringer Ingelheim”, ferroportin mAb and hepcidin mAbfrom Eli Lily, and SelG1 from Selexys Pharmaceuticals, Corp.(“Selexys”).

One or more mAbs in early-stage development for treating various cancersand related conditions can be included in a low-viscosity,high-molecular-weight liquid formulation. United Therapeutics,Corporation has two mAbs in Phase I trials, 8H9 mAb and ch14.18 mAb. ThemAbs ABT-806, enavatuzumab, and volociximab from AbbVie are inearly-stage development. Actinium Pharmaceuticals, Inc has conductedearly-stage trials for the mAbs Actimab-A (M195 mAb), anti-CD45 mAb, andIomab-B. Seattle Genetics, Inc. (“Seattle Genetics”) has several mAbs inearly-stage trials for cancer and related conditions, includinganti-CD22 ADC (RG7593; pinatuzumab vedotin), anti-CD79b ADC (RG7596),anti-STEAP1 ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc.(“Agensys”) the antibody-drug conjugate RG7458, and vorsetuzumabmafodotin. The early-stage cancer therapeutics from Genentech can beincluded in low-viscosity formulations, including ALT-836, theantibody-drug conjugates RG7600 and DEDN6526A, anti-CD22 ADC (RG7593),anti-EGFL7 mAb (RG7414), anti-HER3/EGFR DAF mAb (RG7597), anti-PD-L1 mAb(RG7446), DFRF4539A, an MINT1526A. Bristol-Myers Squibb is developingearly-stage mAbs for cancer therapeutics, including those identified asanti-CXCR4, anti-PD-L1, IL-21 (BMS-982470), lirilumab, and urelumab(anti-CD137). Other mAbs in early-stage trials as cancer therapeuticsinclude APN301(hu14.18-IL2) from Apeiron Biologics AG, AV-203 from AVEO,Pharmaceuticals, Inc. (“AVEO”), AVX701 and AVX901 from AlphaVax, BAX-69from Baxter International, Inc. (“Baxter”), BAY 79-4620 and BAY 20-10112from Bayer HealthCare AG, BHQ880 from Novartis AG,212-Pb-TCMCtrastuzumab from AREVA Med, AbGn-7 from AbGenomicsInternational Inc., and ABIO-0501 (TALL-104) from Abiogen Pharma S.p.A.

Other antibody therapeutics that can be formulated withviscosity-lowering agents include alzumab, GA101, daratumumab,siltuximab, ALX-0061, ALX-0962, ALX-0761, bimagumab (BYM338), CT-011(pidilizumab), actoxumab/bezlotoxumab (MK-3515A), MK-3475(pembrolizumab), dalotuzumab (MK-0646), icrucumab (IMC-18F1, LY3012212),AMG 139 (MEDI2070), SAR339658, dupilumab (REGN668), SAR156597,SAR256212, SAR279356, SAR3419, SAR153192 (REGN421, enoticumab),SAR307746 (nesvacumab), SAR650984, SAR566658, SAR391786, SAR228810,SAR252067, SGN-CD19A, SGN-CD33A, SGN-LIV1A, ASG 15ME, Anti-LINGO,BIIB037, ALXN1007, teprotumumab, concizumab, anrukinzumab (IMA-638),ponezumab (PF-04360365), PF-03446962, PF-06252616, etrolizumab (RG7413),quilizumab, ranibizumab, lampalizumab, onclacumab, gentenerumab,crenezumab (RG7412), IMC-RON8 (narnatumab), tremelimumab, vantictumab,eemcizumab, ozanezumab, mapatumumab, tralokinumab, XmAb5871, XmAb7195,cixutumumab (LY3012217), LY2541546 (blosozumab), olaratumab (LY3012207),MEDI4893, MEDI573, MEDI0639, MEDI3617, MEDI4736, MEDI6469, MEDI0680,MEDI5872, PF-05236812 (AAB-003), PF-05082566, BI 1034020, RG7116,RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652 (MPSK3169A),RG7686, HuMaxTFADC, MOR103, BT061, MOR208, OMP59R5 (anti-notch 2/3),VAY736, MOR202, BAY94-9343, LJM716, OMP52M51, GSK933776, GSK249320,GSK1070806, NN8828, CEP-37250/KHK2804 AGS-16M8F, AGS-16C3F, LY3016859,LY2495655, LY2875358, and LY2812176.

Other early stage mAbs that can be formulated with viscosity-loweringagents include benralizumab, MEDI-8968, anifrolumab, MEDI7183,sifalimumab, MEDI-575, tralokinumab from AstraZeneca and MedImmune;BAN2401 from Biogen Idec/Eisai Co. LTD (“Eisai”)/BioArctic NeuroscienceAB; CDP7657 an anti-CD40L monovalent pegylated Fab antibody fragment,STX-100 an anti-avB6 mAb, BIIB059, Anti-TWEAK (BIIB023), and BIIB022from Biogen; fulranumab from Janssen and Amgen; BI-204/RG7418 fromBioinvent International/Genentech; BT-062 (indatuximab ravtansine) fromBiotest Pharmaceuticals Corporation; XmAb from BoehringerIngelheim/Xencor; anti-IP10 from Bristol-Myers Squibb; J 591 Lu-177 fromBZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX-0401 fromCelldex Therapeutics; foravirumab from Crucell; tigatuzumab from DaiichiSankyo Company Limited; MORAb-004, MORAb-009 (amatuximab) from Eisai;LY2382770 from Eli Lilly; DI17E6 from EMD Serono Inc; zanolimumab fromEmergent BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; catumaxomabfrom Fresenius SE & Co. KGaA; pateclizumab, rontalizumab from Genentech;fresolimumab from Genzyme & Sanofi; GS-6624 (simtuzumab) from Gilead;CNTO-328, bapineuzumab (AAB-001), carlumab, CNTO-136 from Janssen; KB003from KaloBios Pharmaceuticals, Inc.; ASKP1240 from Kyowa; RN-307 fromLabrys Biologics Inc.; ecromeximab from Life Science Pharmaceuticals;LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly; MBL-HCV1 fromMassBiologics; AME-133v from MENTRIK Biotech, LLC; abituzumab from MerckKGaA; MM-121 from Merrimack Pharmaceuticals, Inc.; MCS110, QAX576,QBX258, QGE031 from Novartis AG; HCD122 from Novartis AG and XOMACorporation (“XOMA”); NN8555 from Novo Nordisk; bavituximab, cotara fromPeregrine Pharmaceuticals, Inc.; PSMA-ADC from ProgenicsPharmaceuticals, Inc.; oregovomab from Quest Pharmatech, Inc.; fasinumab(REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331,RG7745 from Roche; ibalizumab (TMB-355) from TaiMed Biologics Inc.;TCN-032 from Theraclone Sciences; TRC105 from TRACON Pharmaceuticals,Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio,Inc.; ABT-110 from AbbVie; Caplacizumab, Ozoralizumab from Ablynx;PRO140 from CytoDyn, Inc.; GS-CDA1, MDX-1388 from Medarex, Inc.; AMG827, AMG 888 from Amgen; ublituximab from TG Therapeutics Inc.; TOL101from Tolera Therapeutics, Inc.; huN901-DM1 (lorvotuzumab mertansine)from ImmunoGen Inc.; epratuzumab Y-90/veltuzumab combination (IMMU-102)from Immunomedics, Inc.; anti-fibrin mAb/3B6/22 Tc-99m from Agenix,Limited; ALD403 from Alder Biopharmaceuticals, Inc.; RN6G/PF-04382923from Pfizer; CG201 from CG Therapeutics, Inc.; KB001-A from KaloBiosPharmaceuticals/Sanofi; KRN-23 from Kyowa; Y-90 hPAM 4 fromImmunomedics, Inc.; Tarextumab from Morphosys AG & OncoMedPharmaceuticals, Inc.; LFG316 from Morphosys AG & Novartis AG; CNTO3157,CNTO6785 from Morphosys AG & Jannsen; RG6013 from Roche & Chugai; MM-111from Merrimack Pharmaceuticals, Inc. (“Merrimack”); GSK2862277 fromGlaxoSmithKline; AMG 282, AMG 172, AMG 595, AMG 745, AMG 761 from Amgen;BVX-20 from Biocon; CT-P19, CT-P24, CT-P25, CT-P26, CT-P27, CT-P4 fromCelltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776Afrom GlaxoSmithKline; anetumab ravtansine from Morphosys AG & Bayer AG;BI-836845 from Morphosys AG & Boehringer Ingelheim; NOV-7, NOV-8 fromMorphosys AG & Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151 fromMerrimack, RG7882 from Roche & Seattle Genetics; RG7841 fromRoche/Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412,PF-05280586 from Pfizer; RG7716, RG7936, gentenerumab, RG7444 fromRoche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212,MEDI-5117, MEDI-7814 from Astrazeneca; ulocuplumab, PCSK9 adnectin fromBristol-Myers Squibb; FPA009, FPA145 from FivePrime Therapeutics, Inc.;GS-5745 from Gilead; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko Kirin;MM-141 from Merck KGaA; REGN1154, REGN1193, REGN1400, REGN1500,REGN1908-1909, REGN2009, REGN2176-3, REGN728 from Regeneron; SAR307746from Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141, ALX-0171 fromAblynx; milatuzumab-DOX, milatuzumab, TF2, from Immunomedics, Inc.;MLN0264 from Millennium; ABT-981 from AbbVie; AbGn-168H from AbGenomicsInternational Inc.; ficlatuzumab from AVEO; BI-505 from BioinventInternational; CDX-1127, CDX-301 from Celldex Therapeutics; CLT-008 fromCellerant Therapeutics Inc.; VGX-100 from Circadian; U3-1565 fromDaiichi Sankyo Company Limited; DKN-01 from Dekkun Corp.; flanvotumab(TYRP1 protein), IL-1 ß antibody, IMC-CS4 from Eli Lilly; VEGFR3 mAb,IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; Anthim from ElusysTherapeutics Inc.; HuL2G7 from Galaxy Biotech LLC; IMGB853, IMGN529 fromImmunoGen Inc.; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaketsuken;KB004 from KaloBios Pharmaceuticals; MGA271, MGAH22 from MacroGenics,Inc.; XmAb5574 from MorphoSys AG/Xencor; ensituximab (NPC-1C) fromNeogenix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 fromNovartis AG; SAN-300 from Santarus Inc.; SelG1 from Selexys; HuM195/rGelfrom Targa Therapeutics, Corp.; VX15 from Teva Pharmaceuticals,Industries Ltd. (“Teva”) and Vaccinex Inc.; TCN-202 from TheracloneSciences; XmAb2513, XmAb5872 from Xencor; XOMA 3AB from XOMA andNational Institute for Allergy and Infectious Diseases; neuroblastomaantibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.;Thravixa from Emergent BioSolutions Inc.; and FB 301 from CytovanceBiologics; rabies mAb from Janssen and Sanofi; flu mAb from Janssen andpartly funded by National Institutes of Health; MB-003 and ZMapp fromMapp Biopharmaceutical, Inc.; and ZMAb from Defyrus Inc.

Other Protein Therapeutics

The protein can be an enzyme, a fusion protein, a stealth or pegylatedprotein, vaccine or otherwise a biologically active protein (or proteinmixture). The term “enzyme,” as used herein, refers to the protein orfunctional fragment thereof that catalyzes a biochemical transformationof a target molecule to a desired product.

Enzymes as drugs have at least two important features, namely i) oftenbind and act on their targets with high affinity and specificity, andii) are catalytic and convert multiple target molecules to the desiredproducts. In certain embodiments, the protein can be PEGylated, asdefined herein.

The term “fusion protein,” as used herein, refers to a protein that iscreated from two different genes encoding for two separate proteins.Fusion proteins are generally produced through recombinant DNAtechniques known to those skilled in the art. Two proteins (or proteinfragments) are fused together covalently and exhibit properties fromboth parent proteins.

There are a number of fusion proteins that are on the market.

ENBREL® (Etanercept), is a fusion protein marketed by Amgen thatcompetitively inhibits TNF.

ELOCTATE®, Antihemophilic Factor (Recombinant), Fc Fusion Protein, is arecombinant DNA derived, antihemophilic factor indicated in adults andchildren with Hemophilia A (congenital Factor VIII deficiency) forcontrol and prevention of bleeding episodes, perioperative management,routine prophylaxis to prevent or reduce the frequency of bleedingepisodes.

EYLEA® (aflibercept) is a recombinant fusion protein consisting ofportions of human VEGF receptors 1 and 2 extracellular domains fused tothe Fc portion of human IgG1 formulated as an iso-osmotic solution forintravitreal administration. EYLEA (aflibercept) is a recombinant fusionprotein consisting of portions of human VEGF receptors 1 and 2extracellular domains fused to the Fc portion of human IgG1 formulatedas an iso-osmotic solution for intravitreal administration. Afliberceptis a dimeric glycoprotein with a protein molecular weight of 97kilodaltons (kDa) and contains glycosylation, constituting an additional15% of the total molecular mass, resulting in a total molecular weightof 115 kDa. Aflibercept is produced in recombinant Chinese hamster ovary(CHO) cells, marketed by Regeneron.

ALPROLIX™, Coagulation Factor IX (Recombinant), Fc Fusion Protein, is arecombinant DNA derived, coagulation Factor IX concentrate is indicatedin adults and children with hemophilia B for control and prevention ofbleeding episodes, perioperative management, routine prophylaxis toprevent or reduce the frequency of bleeding episodes.

Pegloticase (KRYSTEXXA®) is a drug for the treatment of severe,treatment-refractory, chronic gout, developed by SavientPharmaceuticals, Inc. and is the first drug approved for thisindication. Pegloticase is a pegylated recombinant porcine-like uricasewith a molecular weight of about 497 kDa. Pegloticase is currentlyadministered by IV infusions of about 8 mg/kg. High-molecular-weight,low-viscosity liquid formulations can include pegloticase, preferably ina concentration of about 300 mg/mL to about 800 mg/mL.

Alteplase (ACTIVASE®) is a tissue plasminogen activator produced byrecombinant DNA technology. It is a purified glycoprotein comprising 527amino acids and synthesized using the complementary DNA (cDNA) fornatural human tissue-type plasminogen activator obtained from a humanmelanoma cell line. Alteplase is administered via IV infusion of about100 mg immediately following symptoms of a stroke. In some embodiments,low-viscosity formulations are provided containing alteplase, preferablyin a concentration of about 100 mg/mL.

Glucarpidase (VORAXAZE®) is a FDA-approved drug for the treatment ofelevated levels of methotrexate (defined as at least 1 micromol/L)during treatment of cancer patients who have impaired kidney function.Glucarpidase is administered via IV in a single dose of about 50 IU/kg.In some embodiments, low-viscosity formulations are provided containingglucarpidase.

Alglucosidase alfa (LUMIZYME®) is an enzyme replacement therapy orphandrug for treatment of Pompe disease (glycogen storage disease type II),a rare lysosomal storage disorder. It has a molecular weight of about106 kDa and is currently administered by IV infusions of about 20 mg/kg.In some embodiments, a low-viscosity pharmaceutical formulation ofalglucosidase alfa is provided, preferably with a concentration of about100 mg/mL to about 2,000 mg/mL.

Pegdamase bovine (ADAGEN®) is a modified enzyme used for enzymereplacement therapy for the treatment of severe combinedimmunodeficiency disease (SCID) associated with a deficiency ofadenosine deaminase. Pegdamase bovine is a conjugate of numerous strandsof monomethoxypolyethylene glycol (PEG), molecular weight 5,000 Da,covalently attached to adenosine deaminase enzyme that has been derivedfrom bovine intestine.

α-Galactosidase is a lysosomal enzyme that catalyses the hydrolysis ofthe glycolipid, globotriaosylceramide (GL-3), to galactose and ceramidedihexoside. Fabry disease is a rare inheritable lysosomal storagedisease characterized by subnormal enzymatic activity of α-Galactosidaseand resultant accumulation of GL-3. Agalsidase alfa (REPLAGAL®) is ahuman α-galactosidase A enzyme produced by a human cell line. Agalsidasebeta (FABRAZYME®) is a recombinant human α-galactosidase expressed in aCHO cell line. Replagal is administered at a dose of 0.2 mg/kg everyother week by intravenous infusion for the treatment of Fabry diseaseand, off label, for the treatment of Gaucher disease. FABRAZYME® isadministered at a dose of 1.0 mg/kg body weight every other week by IVinfusion. Other lysosomal enzymes can also be used. For example, theprotein can be a lysosomal enzyme as described in US 2012/0148556.

Rasburicase (ELITEK®) is a recombinant urate-oxidase indicated forinitial management of plasma uric acid levels in pediatric and adultpatients with leukemia, lymphoma, and solid tumor malignancies who arereceiving anti-cancer therapy expected to result in tumor lysis andsubsequent elevation of plasma uric acid. ELITEK® is administered bydaily IV infusion at a dosage of 0.2 mg/kg.

Imiglucerase (CEREZYME®) is a recombinant analogue of humanβ-glucocerebrosidase. Initial dosages range from 2.5 U/kg body weight 3times a week to 60 U/kg once every 2 weeks. CEREZYME® is administered byIV infusion.

Abraxane, paclitaxel-conjugated albumin, is approved for metastaticbreast cancer, non-small cell lung cancer, and late stage pancreaticcancer.

Taliglucerase alfa (ELEYSO®) is a hydrolytic lysosomalglucocerebroside-specific enzyme indicated for long-term enzymereplacement therapy for Type 1 Gaucher disease. The recommended dose is60 U/kg of body weight administered once every 2 weeks via intravenousinfusion.

Laronidase (ALDURAZYME®) is a polymorphic variant of the human enzymeα-L-iduronidase that is produced via CHO cell line. The recommendeddosage regimen of ALDURAZYME® is 0.58 mg/kg administered once weekly asan intravenous infusion.

Elosufase alfa (VIMIZIM®) is a human N-acetylgalactosamine-6-sulfataseproduced by CHO cell line by BioMarin Pharmaceuticals Inc (“BioMarin”).It was approved by the FDA on Feb. 14, 2014 for the treatment ofMucopolysaccharidosis Type IVA. It is administered weekly viaintravenous infusion at a dosage of 2 mg/kg.

Other biologics which may be formulated with viscosity-lowering agentsinclude asparaginase erwinia chrysanthemi (ERWINAZE®),incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin Alfa), PROCRIT®(epoetin Alfa), ARANESP® (darbepoetin alfa), ORENCIA® (abatacept),BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE®(Idursulfase); MYOZYME® (LUMIZYME®, algucosidase alfa); VPRIV®(velaglucerase), abobotulinumtoxin A (DYSPORT®); BAX-326, Octocog alfafrom Baxter; Syncria from GlaxoSmithKline; liprotamase from Eli Lilly;Xiaflex (collagenase clostridium histolyticum) from Auxilium andBioSpecifics Technologies Corp.; anakinra from Swedish Orphan BiovitrumAB; metreleptin from Bristol-Myers Squibb; Avonex, Plegridy (BIIB017)from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (darbepoetinalfa), AMG531 (romiplostim), KRN125 (pegfilgrastim), KW-0761(mogamulizumab) from Kyowa; IB1001 from Inspiration Biopharmaceuticals;Iprivask from Canyon Pharmaceuticals Group.

Protein Therapeutics in Development

Versartis, Inc.'s VRS-317 is a recombinant human growth hormone (hGH)fusion protein utilizing the XTEN half-life extension technology. Itaims to reduce the frequency of hGH injections necessary for patientswith hGH deficiency. VRS-317 has completed a Phase II study, comparingits efficacy to daily injections of non-derivatized hGH, with positiveresults. Phase III studies are planned.

Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marinemicroorganism, Vibrio proteolyticus. This endoprotease has specificaffinity for the hydrophobic regions of proteins and is capable ofcleaving proteins adjacent to hydrophobic amino acids. Vibriolysin iscurrently being investigated by Biomarin for the cleaning and/ortreatment of burns. Vibriolysin formulations are described in patent WO02/092014.

PEG-PAL (PEGylated recombinant phenylalanine ammonia lyase or “PAL”) isan investigational enzyme substitution therapy for the treatment ofphenylketonuria (PKU), an inherited metabolic disease caused by adeficiency of the enzyme phenylalanine hydroxylase (PAH). PEG-PAL isbeing developed as a potential treatment for patients whose bloodphenylalanine (Phe) levels are not adequately controlled by KUVAN®.PEG-PAL is now in Phase 2 clinical development to treat patients who donot adequately respond to KUVAN®.

Other protein therapeutics which may be formulated withviscosity-lowering agents include Alprolix/rFIXFc, Eloctate/rFVIIIFc,BMN-190; BMN-250; Lamazyme; Galazyme; ZA-011; Sebelipase alfa; SBC-103;and HGT-1110. Additionally, fusion-proteins containing the XTENhalf-life extension technology including, but not limited to: VRS-317GH-XTEN; Factor VIIa, Factor VIII, Factor IX; PF05280602, VRS-859;Exenatide-XTEN; AMX-256; GLP2-2G/XTEN; and AMX-179 Folate-XTEN-DM1 canbe formulated with viscosity-lowering agents.

Other late-stage protein therapeutics which can be formulated withviscosity-lowering agents include CM-AT from CureMark LLC; NN7999,NN7088, Liraglutide (NN8022), NN9211, Semaglutide (NN9535) from NovoNordisk; AMG 386, Filgrastim from Amgen; CSL-654, Factor VIII from CSLBehring; LA-EP2006 (pegfilgrastim biosimilar) from Novartis AG;Multikine (leukocyte interleukin) from CEL-SCI Corporation; LY2605541,Teriparatide (recombinant PTH 1-34) from Eli Lilly; NU-100 from NuronBiotech, Inc.; Calaspargase Pegol from Sigma-Tau Pharmaceuticals, Inc.;ADI-PEG-20 from Polaris Pharmaceuticals, Inc.; BMN-110, BMN-702 fromBioMarin; NGR-TNF from Molmed S.p.A.; recombinant human C1 esteraseinhibitor from Pharming Group/Santarus Inc.; Somatropin biosimilar fromLG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc.; ART123from Asahi Kasei Corporation; BAX-111 from Baxter; OBI-1 fromInspiration Biopharmaceuticals; Wilate from Octapharma AG; Talactoferrinalfa from Agennix AG; Desmoteplase from Lundbeck; Cinryze from Shire;RG7421 and Roche and Exelixis, Inc.; Midostaurin (PKC412) from NovartisAG; Damoctocog alfa pegol, BAY 86-6150, BAY 94-9027 from Bayer AG;Peginterferon lambda-1a, Nulojix (Belatacept) from Bristol-Myers Squibb;Pergoveris, Corifollitropin alfa (MK-8962) from Merck KGaA; recombinantcoagulation Factor IX Fc fusion protein (rFIXFc; BIIB029) andrecombinant coagulation Factor VIII Fc fusion protein (rFVIIIFc;BIIB031) from Biogen; and Myalept from AstraZeneca.

Other early stage protein biologics which can be formulated withviscosity-lowering agents include Alferon LDO from Hemispherx BioPharma,Inc.; SL-401 from Stemline Therapeutics, Inc.; PRX-102 from ProtalixBiotherapeutics, Inc.; KTP-001 from Kaketsuken/Teijin Pharma Limited;Vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001 (PF-05236806)from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924 from NovoNordisk; INGAP peptide from Exsulin; ABT-122 from Abbvie; AZD9412 fromAstraZeneca; NEUBLASTIN (BG00010) from Biogen; Luspatercept (ACE-536),Sotatercept (ACE-011) from Celgene Corporation; PRAME immunotherapeuticfrom GlaxoSmithKline; Plovamer acetate (PI-2301) from Merck KGaA;PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Ontak from Eisai;rHuPH20/insulin from Halozyme, Inc.; PB-1023 from PhaseBioPharmaceuticals, Inc.; ALV-003 from Alvine Pharmaceuticals Inc. andAbbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon TherapeuticsInc.; PEGPH20 from Halozyme, Inc.; Amevive® alefacept from AstellasPharma Inc.; F-627 from Regeneron; AGN-214868 (senrebotase) fromAllergan, Inc.; BAX-817 from Baxter; PRT4445 from PortolaPharmaceuticals, Inc.; VEN100 from Ventria Bioscience;Onconase/ranpirnase from Tamir Biotechnology Inc.; interferon alpha-2binfusion from Medtronic, Inc; sebelipase alfa from Synageva BioPharma;IRX-2 from IRX Therapeutics, Inc; GSK2586881 from GlaxoSmithKline;SI-6603 from Seikagaku Corporation; ALXN1101, asfotase alfa fromAlexion; SHP611, SHP609 (Elaprase, idursulfase) from Shire; PF-04856884,PF-05280602 from Pfizer; ACE-031, Dalantercept from Acceleron Pharma;ALT-801 from Altor BioScience Corp.; BA-210 from BioAxone Biosciences,Inc.; WT1 immunotherapeutic from GlaxoSmithKline; GZ402666 from Sanofi;MSB0010445, Atacicept from Merck KGaA; Leukine (sargramostim) from BayerAG; KUR-211 from Baxter; fibroblast growth factor-1 from CardioVascularBioTherapeutics Inc.; SPI-2012 from Hanmi Pharmaceuticals Co.,LTD/Spectrum Pharmaceuticals; FGF-18 (sprifermin) from Merck KGaA;MK-1293 from Merck; interferon-alpha-2b from HanAll Biopharma; CYT107from Cytheris SA; RT001 from Revance Therapeutics, Inc.; MEDI6012 fromAztraZeneca; E2609 from Biogen; BMN-190, BMN-270 from BioMarin; ACE-661from Acceleron Pharma; AMG 876 from Amgen; GSK3052230 fromGlaxoSmithKline; RG7813 from Roche; SAR342434, Lantus from Sanofi; AZ01from Allozyne Inc.; ARX424 from Ambrx, Inc.; FP-1040, FP-1039 fromFivePrime Therapeutics, Inc.; ATX-MS-1467 from Merck KGaA; XTEN fusionproteins from Amunix Operating Inc.; entolimod (CBLB502) from ClevelandBioLabs, Inc.; HGT2310 from Shire; HM10760A from Hanmi PharmaceuticalsCo., LTD; ALXN1102/ALXN1103 from Alexion; CSL-689, CSL-627 from CSLBehring; glial growth factor 2 from Acorda Therapeutics, Inc.; NX001from Nephrx Corporation; NN8640, NN1436, NN1953, NN9926, NN9927, NN9928from Novo Nordisk; NHS-IL 12 from EMD Serono; 3K3A-APC from ZZ BiotechLLC; PB-1046 from PhaseBio Pharmaceuticals, Inc.; RU-101 from R-TechUeno, Ltd.; insulin lispro/BC106 from Adocia; hl-con1 from IconicTherapeutics, Inc.; PRT-105 from Protalix BioTherapeutics, Inc.;PF-04856883, CVX-096 from Pfizer; ACP-501 from AlphaCore Pharma LLC;BAX-855 from Baxter; CDX-1135 from Celldex Therapeutics; PRM-151 fromPromedior, Inc.; TS01 from Thrombolytic Science International; TT-173from Thrombotargets Corp.; QBI-139 from Quintessence Biosciences, Inc.;Vatelizumab, GBR500, GBR600, GBR830, and GBR900 from GlenmarkPharmaceuticals; and CYT-6091 from Cytimmune Sciences, Inc.

Other Biologic Agents

Other biologic drugs that can be formulated with viscosity-loweringagents include PF-05285401, PF-05231023, RN317 (PF-05335810),PF-06263507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598,RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMC-3G3, IMC-18F1,IMC-35C, IMC-20D7S, PF-06480605, PF-06647263, PF-06650808, PF-05335810(RN317) PD-0360324, PF-00547659 from Pfizer; MK-8237 from Merck; BI033from Biogen; GZ402665, SAR438584/REGN2222 from Sanofi; IMC-18F1; andIcrucumab, IMC-3G3 from ImClone LLC; Ryzodeg, Tresiba, Xultophy fromNovo Nordisk; Toujeo (U300), LixiLan, Lyxumia (lixisenatide) fromSanofi; MAGE-A3 immunotherapeutic from GlaxoSmithKline; Tecemotide fromMerck KGaA; Sereleaxin (RLX030) from Novartis AG; Erythropoietin;Pegfilgrastim; LY2963016, Dulaglutide (LY2182965) from Eli Lilly; andInsulin Glargine from Boehringer Ingelheim.

B. Viscosity-Lowering Agents

The viscosity of liquid protein formulations, includinglow-molecular-weight and/or high-molecular-weight proteins, is reducedby the addition of one or more viscosity-lowering agents. Thepharmaceutical formulations may be converted from non-Newtonian toNewtonian fluids by the addition of an effective amount of one or moreviscosity-lowering agents.

When employed in a formulation intended for administration to a human orother mammal, the viscosity-lowering agents, like the formulationitself, must be pharmaceutically acceptable. The viscosity-loweringagents are typically organic compounds containing at least onenon-carbon, non-hydrogen atom. Preferably, the viscosity-lowering agentscontain hydrogen, carbon, oxygen and at least one other type of atom. Incertain embodiments, the viscosity-lowering agents are characterized byat least one of the following:

-   -   1) organic compounds having at least four carbon and four        hydrogen atoms, and at least one sulfur, oxygen, nitrogen, or        phosphorus atom;    -   2) a molecular weight between about 85 and 1,000 Da;    -   3) the presence of at least one charged, or other hydrophilic,        moiety;    -   4) the presence of at least one, preferably two, and more        preferably three, freely rotating bonds;    -   5) the presence of at least one substituted ring;    -   6) a molecular polar surface area of at least 24 Å², preferably        at least 50 Å², and more preferably at least 80 Å²;    -   7) a molar volume of at least 75 cm³, preferably at least 85        cm³, more preferably at least 100 cm³, and most preferably at        least 120 cm³;    -   8) a polarizability of at least 10 cm³, preferably at least 15        cm³, more preferably at least 20 cm³, and most preferably at        least 25 cm³; and    -   9) the presence of at least one, preferably two, and more        preferably three hydrogen bond donors and/or acceptors.

In certain embodiments, the viscosity-lowering agent is characterized byat least two, three, four, five, six, seven, eight or all nine of theabove listed attributes. In certain embodiments, the viscosity-loweringagent is further characterized in that it does not contain an aldehydeor carbon-carbon triple bond functional group.

In other embodiments, the viscosity-lowering agent is a combination oftwo or more compounds, each of which is characterized by at least two,three, four, five, six, seven, eight or all nine of the above listedattributes.

In some embodiments, the viscosity-lowering agents are listed as GRAS bythe U.S. Food and Drug Administration (“the FDA”), as of Sep. 11, 2014.“GRAS” is an acronym for the phrase Generally Recognized As Safe. Undersections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act (theAct), any substance that is intentionally added to food is a foodadditive and is subject to premarket review and approval by FDA unlessthe substance is generally recognized, among qualified experts, ashaving been adequately shown to be safe under the conditions of itsintended use, or unless the use of the substance is otherwise excludedfrom the definition of a food additive. Another source of compounds isthe Inactive Ingredient Guide of the FDA (IIG), and equivalents listedby the International Pharmaceutical Excipients Council (IPEC) and theEuropean Medicines Agency (EMA), as of Sep. 11, 2014. The substancesused in formulations must be safe for injection. Preferably, theGRAS-listed viscosity-lowering agent is characterized by at least two,three, four, five, six, seven, eight or all nine of the above listedattributes.

In other embodiments, the viscosity-lowering agent is an FDA- orEMA-approved drug product as of Sep. 11, 2014. Like compounds drawn fromthe GRAS and IIG lists, the toxicity and safety profiles of FDA- andEMA-approved drug products are well established. In addition to loweringthe viscosity of the protein solution, the use of an FDA- orEMA-approved drug product provides the opportunity for combinationtherapies. Preferably a FDA- or EMA-approved drug productviscosity-lowering agent is characterized by at least two, three, four,five, six, seven, eight or all nine of the above listed attributes.

In some embodiments, the viscosity-lowering agent includes at least onecompound of Formula (I):

or a pharmaceutically acceptable salt thereof;wherein

represents either a single or double bond, A is a selected from O, S,SO₂, NR³, C(R³)₂ or:

wherein R³ is independently selected from hydrogen, R², —OH, NH₂, —F,—Cl, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —PO₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a),and —N(R^(4a))₂;

wherein R² is independently selected from C₁₋₁₂alkyl, C₃₋₁₂cycloalkyl,C₆₋₁₂aryl, C₁₋₁₂heteroaryl and C₂₋₁₂heterocyclyl;

wherein each C₁₋₁₂alkyl may be substituted one or more times withC₃₋₁₂cycloalkyl, C₆₋₁₂aryl, C₁₋₁₂heteroaryl, C₂₋₁₂heterocyclyl, —OH,NH₂, (═O), (═NR^(4a)), —F, —Cl, —Br, —I, —NO₂, —CN, —C(═O)R^(4a),—C(═NR^(4a))R⁴, —C(═O)OH, —C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H,—SO₂N(R^(4a))₂, —SO₂R⁴, —SO₂NR^(4a)C(═O)R⁴, —PO₃H₂,—R^(4a)C(═NR^(4a))N(R^(4a))₂, —NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴,—NR^(4a)SO₂R⁴, —NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or—N(R^(4a))₂;

wherein each C₃₋₁₂cycloalkyl may be substituted one or more times withC₁₋₁₂alkyl, C₆₋₁₂aryl, C₁₋₁₂heteroaryl, C₂₋₁₂heterocyclyl, —OH, NH₂, —F,—Cl, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —P₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or—N(R^(4a))₂;

wherein each C₆₋₁₂aryl may be substituted one or more times withC₁₋₁₂alkyl, C₃₋₁₂cycloalkyl, C₁₋₁₂heteroaryl, C₂₋₁₂heterocyclyl, —OH,NH₂, —F, —C, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —PO₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or—N(R^(4a))₂;

wherein each C₁₋₁₂heteroaryl may be substituted one or more times withC₁₋₁₂alkyl, C₃₋₁₂cycloalkyl, C₆₋₁₂aryl, C₂₋₁₂heterocyclyl, —OH, NH₂, —F,—C, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —PO₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or—N(R^(4a))₂;

wherein each C₂₋₁₂heterocyclyl may be substituted one or more times withC₁₋₁₂alkyl, C₃₋₁₂cycloalkyl, C₆₋₁₂aryl, C₁₋₁₂heteroaryl, —OH, NH₂, —F,—C, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —PO₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or—N(R^(4a))₂;

wherein R⁴ is independently selected from C₁₋₁₂alkyl, C₃₋₁₂cycloalkyl,C₆₋₁₂aryl, C₁₋₁₂heteroaryl and C₂₋₁₂heterocyclyl, each of which may besubstituted one or more times by —OH, —NH₂, —F, —Cl, —Br, —I, —NO₂, —CN,—C(═O)OH, —SO₃H, —PO₃H₂, or —C(═O)NH₂;

wherein R^(4a) may be R⁴ or hydrogen;

wherein any two or more of R², R³, R⁴ and R^(4a) groups may togetherform a ring;

wherein when two R³ groups are bonded to the same carbon atom, the twoR³ groups may together form an (═O), (═NR^(4a)), or (═C(R^(4a))₂);

wherein z is in each case independently selected from 1 or 2, providedthat when the (R³)_(z) substituent is connected to an sp² hybridizedcarbon, z is 1, and when the (R³)_(z) substituent is connected to an sp³hybridized carbon, z is 2.

When the substituent —NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂ ispresent, it is preferred that R^(4a) is selected so as to give—NHC(═NH)NHC(═NH)NH₂.

In certain embodiments, the compound of Formula (1) contains at leastone substituent selected from —C(═O)OH, —SO₃H, —SO₂NHC(═O)R⁴, and—PO₃H₂. In some embodiments, the compound of Formula (1) contains atleast one —SO₃H group.

In certain embodiments, one or more of the R³ substituents may be:

wherein R^(3a) and R^(3b) are independently selected from hydrogen,C₁₋₁₂alkyl, C₃₋₁₂cycloalkyl, C₆₋₁₂aryl, C₁₋₁₂heteroaryl andC₂₋₁₂heterocyclyl, C(═O)R^(4a), —C(═O)OH, —C(═O)OR⁴, —SO₃H,—SO₂N(R^(4a))₂, —SO₂R⁴, —SO₂NHC(═O)R⁴, C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴,—SR⁴, and —N(R^(4a))₂, and when any two R^(3b) are bonded to the samecarbon atom, the two R^(3b) groups may together form an (═O),(═NR^(4a)), or (═C(R^(4a))₂);

wherein each C₁₋₁₂alkyl, C₃₋₁₂cycloalkyl, C₆₋₁₂aryl, C₁₋₁₂heteroaryl andC₂₋₁₂heterocyclyl may be substituted one or more times with —OH, NH₂,—F, —Cl, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —PO₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂,—NR^(4a)C(═O)N(R^(4a))₂, —C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or—N(R^(4a))₂;

wherein R⁴ and R^(4a) are as defined above;

wherein x is selected from 1, 2, 3, 4, 5, 7, 8, 9 or 10; and

wherein any two or more of R³, R^(3a), R⁴ and R^(4a) groups may togetherform a ring.

In certain embodiments, the compound of Formula (1) may be representedby either the compound of Formula (1a) or (1b):

wherein R³ has the meanings given above.

In certain embodiments, the compound of Formula (1a) may be representedby the compounds of Formulas (1 a-i-iv):

wherein R³ is independently selected from hydrogen, NH₂, CH₃, Cl, OR⁴and NHR⁴;

wherein x is 1 or 2;

wherein R^(3a) and R^(3b) are independently selected from hydrogen andC₁₋₁₂ alkyl;

wherein said C₁₋₁₂alkyl may be substituted one or more times by C₃₋₁₂cycloalkyl, C₆₋₁₂aryl, C₁₋₁₂heteroaryl, C₂₋₁₂heterocyclyl, —OH, NH₂, —F,—Cl, —Br, —I, —NO₂, —CN, —C(═O)R^(4a), —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —OC(═O)R⁴, —OC(═O)OR⁴, —SO₃H, —SO₂N(R^(4a))₂, —SO₂R⁴,—SO₂NR^(4a)C(═O)R⁴, —PO₃H₂, —R^(4a)C(═NR^(4a))N(R^(4a))₂,—NHC(═NR^(4a))NH—CN, —NR^(4a)C(═O)R⁴, —NR^(4a)SO₂R⁴,—NR^(4a)C(═NR^(4a))NR^(4a)C(═NR^(4a))N(R^(4a))₂, NR^(4a)C(═O)N(R^(4a))₂,—C(═O)NH₂, —C(═O)N(R^(4a))₂, —OR⁴, —SR^(4a), or —N(R^(4a))₂;

R⁴ and R^(4a) are as defined above; and

wherein any two or more R^(3a), R^(3b), R⁴, R^(4a) may together form aring.

The compound of Formula (1) may be represented by the compound ofFormula (1a-v, vi or vii):

wherein R^(3f) is selected from —C(═O)OH, —SO₃H, —SO₂NHC(═O)R⁴, and—PO₃H₂, and R³ is as defined above. In certain preferred embodiments, R³is independently selected from hydrogen, OH, NH₂, C₁₋₆alkyl and COOH.

In other embodiments, the compound of Formula (1) may be represented byany of the compounds of Formulae (1c), (1d), (1e) or (1f):

wherein R³ has the meanings given above.

In other embodiments, the compound of Formula (1) may be represented bya compound of Formula (1g):

wherein R^(3c) is independently selected from hydrogen and R², whereinR² has the meanings given above;

wherein R^(3d) is independently selected from hydrogen, OH, NH₂,NH(C₁₋₆alkyl), N(C₁₋₆alkyl)₂; NHC(═O)(C₁₋₆alkyl), COOH and CH₂OH;

or any two R^(3c) and R^(3d) groups connected to the same carbon maytogether form an oxo (═O), imino (═NR^(4a)), or an olefin (═C(R^(4a))₂),wherein R^(4a) has the meanings given above;

wherein R^(3e) is selected from hydrogen, —OH or OR⁴; and

wherein R⁴ has the meanings given above.

In certain embodiments, the viscosity-lowering agent includes a compoundof Formula (1g-i):

wherein R^(3e) is selected from OH and —OC₁₋₁₂alkyl, which is furthersubstituted with at least one OH and at least one COOH; and

wherein R^(3d) is selected from COOH and CH₂OH.

In some embodiments, the viscosity-lowering agent includes a compound ofFormula (2):

or a pharmaceutically acceptable salt thereof;

wherein

represents a single or double bond;

X is independently selected from chalcogen, N(R³)_(z) and C(R³)_(z);

X¹ is absent, or is chalcogen, N(R³)_(z), C(R³)_(z) or:

wherein R³ has the meanings given for the compound of Formula (1);provided that when the (R³)_(z) substituent is connected to an sp²hybridized nitrogen, z is 0 or 1, when the (R³)_(z) substituent isconnected to an sp² hybridized carbon or an sp³ hybridized nitrogen, zis 1, and when the (R³)_(z) substituent is connected to an sp³hybridized carbon, z is 2;

wherein at least one of X or X¹ is chalcogen or N(R³)_(z).

In certain embodiments, the compound may be an aromatic ring. Exemplaryaromatic rings include the compounds of Formulas (2a-e):

wherein R³ and X have the meanings above, and X² is selected fromN(R³)_(z) and C(R³)_(z).

In certain embodiments, the viscosity-lowering agent is a compound ofFormula (2a-i):

wherein R⁴ is as defined above and is preferably hydrogen or CH₃;

wherein R⁶ is C₁₋₁₂heteroaryl, which may be substituted one or moretimes by C₁₋₆alkyl;

wherein said C₁₋₆alkyl may be substituted one or more times by OH, —NH₂,—F, —Cl, —Br, —I, —NO₂, —CN, —C(═O)R⁴, —C(═NR^(4a))R⁴, —C(═O)OH,—C(═O)OR⁴, —SO₃H, —SO₂NR⁴—, —SO₂R⁴, —PO₃H₂, —NHC(═O)R⁴, —NHC(═O)N(R⁴)₂,—C(═O)NH₂, —C(═O)N(R⁴)₂, —OR^(4b), —SR^(4b), —N(R^(4b))₂, wherein R⁴ hasthe meanings given above; or

wherein R⁴ is as defined above, and R⁷ is selected from SR⁴ and—C(═O)R⁴. The double bond in the group above may be in either the E or Zgeometry.

In preferred embodiments, R⁶ is a heterocycle having the structure:

wherein X⁴ is a chalcogen and R^(6a) is hydrogen or C₁₋₆alkyl, whereinthe C₁₋₆alkyl may be substituted one or more times by —OH, —NH₂, —F,—Cl, —Br, —I, —NO₂, —CN, —C(═O)OH. In an even more preferred embodiment,R⁶ is a heterocycle having the structure:

wherein R^(6a) is selected from unsubstituted C₁₋₆alkyl and C₁₋₆alkylsubstituted one or more times with —OH.

The viscosity-lowering agent may be an imidazole of Formula (2b-i)

wherein R³ is as defined above. In certain embodiments, R³ isindependently selected from hydrogen, NO₂, and R⁴. In certain preferredembodiments, the compound of Formula (2b-i) has the structure:

wherein R³ is independently selected from C₁₋₆ alkyl, which may beunsubstituted or substituted one or more times with a group selectedfrom OH, NH₂, SR⁴, F, Cl, Br and I; and

R^(3g) is either hydrogen or NO₂.

In other embodiments, the viscosity-lowering agent has the structure ofFormula (2a-ii) or Formula (2c-i):

wherein R³ is independently selected from OH, Cl, Br, F, I, N(R^(4a))₂,C(═O)OH, C(═O)NH₂.

In further embodiments, at least one R³ substituent is NHR⁴, wherein R⁴is a C₁₋₆alkyl, optionally substituted by one or more groups selectedfrom Cl, Br, F, I, OH, C(═O)OH, NH₂, NH(C₁₋₆alkyl) and N(C₁₋₆alkyl)₂.

In other embodiments, the viscosity-lowering agent is a pyridinium saltof Formula (2a-iii):

wherein R³ and R⁴ are as defined above.

In other embodiments, the heterocyclic ring is not a heteroaryl ring.Exemplary non-aromatic rings include the compounds of Formulas (2f-k):

wherein R⁵ and X have the meanings above, and X³ is chalcogen orN(R³)_(z).

In certain embodiments, the compound of Formula (2f) is a beta-lactam ofFormula (2f-i),

The beta lactam of Formula (2f-i) includes penicillin-type compounds, aswell cephalosporin-type and cephamycin-type compounds of the Formula(2f-ii) and (2f-iii):

wherein X and R3 are as defined above. In preferred embodiments, X issulfur.

In certain embodiments, the compound of Formula (2i) is a compound ofFormula (2i-i):

wherein X and R³ are as defined above. In certain embodiments, X is inboth cases NR⁴, wherein R⁴ has the meanings given above, and R³ is inboth cases hydrogen.

In other embodiments, the compound of Formula (2) is represented by acompound of Formula (2i-ii):

wherein X, X¹ and R³ are as defined above.

The compound of Formula (2j) may be represented by the compound ofFormula (2j-i):

wherein X³ and R³ are as defined above, and R⁸ is selected from theNHC(═O)R² and OC(═O)R². In preferred embodiments, X³ is N⁺(CH₃)₂, R³ areboth hydrogen, or R³ together form an epoxide or double bond.

The compound of Formula (2k) may be represented by the compound ofFormula (2k-i):

wherein X³ and R⁸ are as defined above.

In other embodiments, the viscosity-lowering agent includes a compoundof the structure of Formula (3):

or a pharmaceutically acceptable salt thereof;wherein R⁵ is in each case independently selected from hydrogen, and R²,

R^(5′) is either R⁵ or absent;

providing that at least one R⁵ substituent is not hydrogen, wherein R²has the same meanings given for the compound or Formula (1).

In certain embodiments, the viscosity-lowering agent is a mixture of twoor more compounds selected from compounds of Formula (1), Formula (2)and Formula (3).

In preferred embodiments, the viscosity-lowering agent iscamphorsulfonic acid (CSA), or a pharmaceutically acceptable saltsthereof, such as an alkaline or alkaline earth metal salt. Thecamphorsulfonic acid or salt thereof is combined with one or morecompounds of Formula (1), (2) or (3) to give mixtures such asCSA-piperazine, CSA-TRIS, CSA-4-amino pyridine,CSA-1-(o-tolyl)biguanide, CSA-procaine, CSA-Na-aminocyclohexanecarboxylic acid, CSA-Na-creatinine and CSA-Na-ornidazole. Otherpreferred viscosity-lowering agents include thiamine, procaine, biotin,creatinine, metoclopramide, scopolamine, cimetidine, chloroquinephosphate, mepivacaine, granisetron, sucralose, HEPES-tris,nicotinamide, lactobionic acid-TRIS, glucuronic acid-TRIS,sulfacetamide, CSA-4-aminopyridine, CSA-piperazine and cefazolin. Anytwo or more of the viscosity-lowering agents listed above may further becombined in the same formulation.

In other embodiments, the viscosity-lowering agent is an organosulfonicacid. Exemplary organosulfonic acids include, but are not limited to,camphorsulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid,toluenesulfonic acid, cyclohexylsuflonic acid, xylenesulfonic acids(including p-xylene-2-sulfonic acid, m-xylene-2-sulfonic acid,m-xylene-4-sulfonic acid and o-xylene-3-sulfonic acid), methanesulfonicacid, 1,2 ethane disulfonic acid, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 2-hydroxyethane-1-sulfonic acid,3-hydroxypropane-1-sulfonic acid, cymenesulfonic acid,4-hydroxybutane-1-sulfonic acid and pharmaceutically acceptable saltsthereof. The organosulfonic acid may be in the form of an alkaline oralkaline earth metal salt, such as lithium, sodium, potassium,magnesium, and calcium salt. The organosulfonic acid (or salt thereof)may be combined with one or more compounds of Formula (2) or Formula(3).

In certain embodiments, the viscosity-lowering agent contains at leastone carboxylic acid. The carboxylic acid may be in the form of analkaline or alkaline earth metal salt, such as lithium, sodium,potassium, magnesium, and calcium salt. Exemplary carboxylic acidcompounds include lactobionic acid, glucuronic acid, 1-aminocyclohexanecarboxylic acid, biotin, brocrinat, cyclopentane propionic acid,hydroxynaphthoic acid, phenylpropionic acid, gentisic acid, salicylicacid, camphoric acid, mandelic acid, sulfosalicyclic acid,hydroxybenzoyl benzoic acid, phenyl acetic acid, acetyl salicylic acid,cinnamic acid, t-butyl acetic acid, phthalic acid, trimethylacetic acid,anthrallic acid and pharmaceutically acceptable salts thereof. Thecarboxylic acid (or salt thereof) may be combined with one or morecompounds of Formula (2) or Formula (3).

The following compounds may also be used as viscosity-lowering agents:colistin, articaine, tetracaine, proxymetacaine, metoclopramide,procaine, lidocaine, cyclomethylcaine, piperocaine, chloroprocaine,etidocaine, benzocaine, phenylephrine, bupivacaine, mepivacaine,cinchocaine, mixtures thereof and pharmaceutically acceptable saltsthereof.

Other agents which may be employed as viscosity-lowering agents include1-aminocyclohexane carboxylic acid, 1-(o-tolyl)biguanide, benzethoniumchloride, benzoic acid, brocrinat, calcium carrageenan, calciumcyclamate, calcobutrol, caloxetic acid, camphorsulfonic acid,creatinine, dalfampridine, dehydroacetic acid, diazolidinyl urea,dichlorobenzyl alcohol, dimethyl isosorbide, epitetracycline, ethylmaltol, ethyl vanillin, ornidazole, gentisic acid ethanolamide, HEPES(4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid), gentisic acid,glucuronic acid, iodoxamic acid, menthol, galactose, medronic acid,m-cresol, glutathione, lactobionic acid, maltitol, octisalate,oxyquinoline, pentetic acid, piperazine, propenyl guaethol, propylgallate, propylene carbonate, propylparaben, protamine sulfate,QUATERNIUM-15, QUATERNIUM-52, satialgine H, sodium1,2-ethanedisulfonate, sodium cocoyl sarcosinate, sodium lauroylsarcosinate, sodium polymetaphosphate, sodium pyrophosphate,pyroglutamic acid, sodium trimetaphosphate, sodium tripolyphosphate,sorbitan, tartaric acid, lactic acid, iofetamine, sucralose,1-(4-pyridyl)pyridinium chloride, aminobenzoic acid, sulfacetamidesodium, naphthalene-2-sulfonic acid, tert-butylhydroquinone, thimerosal,trolamine, tromantadine, vanillin, versetamide, nioxime, niacinamide,methylisothiazolinone, mannose D, maltose, lidofenin, lactose, lactitol,isomalt, imidurea, gluconolactone, methanesulfonic acid, xylenesulfonicacid, sulfobutylether β-cyclodextrin and pharmaceutically acceptablesalts thereof.

In certain embodiments, the viscosity-lowering agent includes an organicbase. Exemplary organic bases include N-methylglucamine, morpholine,piperidine, and primary, secondary, tertiary, and quaternary amines,substituted amines, and cyclic amines. For example, they can beisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,lidocaine, hydrabamine, cholines, betaines, choline, betaine,ethylenediamine, theobromine, purines, piperazine, N-ethylpiperidine,N-methylpiperidinepolyamine. Particularly preferred organic bases arearginine, histidine, lysine, ethanolamine, thiamine,2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), 4-aminopyridine,aminocyclohexane carboxylic acid, 1-o-tolybiguanide, ornidazole, urea,nictoinamide, benzethonium chloride, 5-amino-1-pentanol,2-(2-aminoethoxy)ethanol, trans-cyclohexane-1,4-diamine,trans-cyclohexane-1R,2R-diamine, ethylenediamine, propane-1,3-diamine,butane-1,4-diamine, pentane-1,5-diamine, hexane-1,6-diamine,octane-1,8-diamine, 5-amino-1-pentanol, 2-(2-aminoethoxy)ethanamine,2-(2-(2-aminoethoxy)-ethoxy)ethanamine,3-(4-(3-aminopropoxy)-butoxy)propan-1-amine,3-(2-(2-(3-aminopropoxy)-ethoxy)-ethoxy)propan-1-amine,N-(2-(2-aminoethylamino)ethyl)ethane-1,2-diamine,N-(2-aminoethyl)ethane-1,2-diamine,N-1-(2-(2-(2-aminoethylamino)ethylamino)-ethyl)ethane-1,2-diamine,N,N-dimethylhexane-1,6-diamine, N,N,N,N-tetramethylbutane-1,4-diamine,phenyltrimethylammonium salts, isopropylamine, diethylamine,ethanolamine, trimethamine, choline,1-(3-aminopropyl)-2-methyl-1H-imidazole, piperazine,1-(2-aminoethyl)piperazine, 1-[3-(dimethylamino)propyl]piperazine,1-(2-aminoethyl)piperidine, 2-(2-aminoethyl-1-methylpyrrolidine,mixtures thereof, and pharmaceutically acceptable salts thereof.

Exemplary beta-lactams include benzylpenicillin (penicillin G),phenoxymethylpenicillin (penicillin V), cloxacillin, dicloxacillin,flucloxacillin, methicillin, nafcillin, oxacillin, temocillin,amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin,azlocillin, mezlocillin, piperacillin, cefoxitin, cefazolin, cephalexin,cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime,cefotetan, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone,cefepime, cefpirome, ceftobiprole, biapenem, doripenem, ertapenemfaropenem, imipenem, meropenem, panipenem, razupenem, tebipenem,thienamycin, aztreonam, tigemonam, nocardicin a, tabtoxinine, clavulanicacid, clavulanic acid, tazobactam, sulbactam and pharmaceuticallyacceptable salts thereof.

Other viscosity-lowering agents include tropane N-heterocycles, such asatropine, hyoscyamine, scopolamine, and salts thereof, as well astiotropium and ipratropium salts, thiamine, allithiamine, prosultiamine,fursultiamine, benfotiamine, sulbutiamine, quaternium 15;1-(3-aminopropyl)-2-methyl-1H-imidazole dihydrochloride; creatinine;biotin, cimetidine, piperocaine, cyclomethylcaine, granisetron,moxifloxacin, chloroquine, mepivacaine, levetriacetam, bupivacaine,cinchocaine, clindamycin and pharmaceutically acceptable salts thereof.Thiamine is an especially preferred viscosity-lowering agent.

In certain formulations, the following compounds are not preferred:creatinine, cadaverine, lidocaine, arginine and lysine, and are excludedfrom the scope of the foregoing formulas and definitions of usefulviscosity-lowering agents.

C. Excipients

A wide variety of pharmaceutical excipients useful for liquid proteinformulations are known to those skilled in the art. They include one ormore additives, such as liquid solvents or co-solvents; sugars or sugaralcohols such as mannitol, trehalose, sucrose, sorbitol, fructose,maltose, lactose, or dextrans; surfactants such as TWEEN® 20, 60, or 80(polysorbate 20, 60, or 80); buffering agents; preservatives such asbenzalkonium chloride, benzethonium chloride, tertiary ammonium salts,and chlorhexidinediacetate; carriers such as poly(ethylene glycol)(PEG); antioxidants such as ascorbic acid, sodium metabisulfite, andmethionine; chelating agents such as EDTA or citric acid; orbiodegradable polymers such as water soluble polyesters;cryoprotectants; lyoprotectants; bulking agents; and stabilizing agents.

Other pharmaceutically acceptable carriers, excipients, or stabilizers,such as those described in Remington: “The Science and Practice ofPharmacy”, 20th edition, Alfonso R. Gennaro, Ed., Lippincott Williams &Wilkins (2000) may also be included in a protein formulation describedherein, provided that they do not adversely affect the desiredcharacteristics of the formulation.

The viscosity-lowering agents described herein can be combined with oneor more other types of viscosity-lowering agents, for example,organophosphates described in co-filed PCT application entitled “LIQUIDPROTEIN FORMULATIONS CONTAINING ORGANOPHOSPHATES” by Arsia Therapeutics;water soluble dyes described in co-filed PCT application entitled“LIQUID PROTEIN FORMULATIONS CONTAINING WATER SOLUBLE ORGANIC DYES” byArsia Therapeutics; ionic liquids described in co-filed PCT applicationentitled “LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS” by ArsiaTherapeutics.

III. Methods of Making

The protein, such as a mAb, to be formulated may be produced by anyknown technique, such as by culturing cells transformed or transfectedwith a vector containing one or more nucleic acid sequences encoding theprotein, as is well known in the art, or through synthetic techniques(such as recombinant techniques and peptide synthesis or a combinationof these techniques), or may be isolated from an endogenous source ofthe protein.

Purification of the protein to be formulated may be conducted by anysuitable technique known in the art, such as, for example, ethanol orammonium sulfate precipitation, reverse phase HPLC, chromatography onsilica or cation-exchange resin (e.g., DEAE-cellulose), dialysis,chromatofocusing, gel filtration using protein A SEPHAROSE® columns(e.g., SEPHADEX® G-75) to remove contaminants, metal chelating columnsto bind epitope-tagged forms, and ultrafiltration/diafiltration(non-limiting examples include centrifugal filtration and tangentialflow filtration (TFF)).

Inclusion of viscosity-lowering agents at viscosity-reducingconcentrations such as 0.010 M to 1.0 M, preferably 0.050 M to 0.50 M,most preferably 0.10 M to 0.30 M, allows a solution of thepharmaceutically active mAb to be purified and/or concentrated at highermAb concentrations using common methods known to those skilled in theart, including but not limited to tangential flow filtration,centrifugal concentration, and dialysis.

In some embodiments, lyophilized formulations of the proteins areprovided and/or are used in the preparation and manufacture of thelow-viscosity, concentrated protein formulations. In some embodiments,the pre-lyophilized protein in a powder form is reconstituted bydissolution in an aqueous solution. In this embodiment, the liquidformulation is filled into a specific dosage unit container such as avial or pre-filled mixing syringe, lyophilized, optionally withlyoprotectants, preservatives, antioxidants, and other typicalpharmaceutically acceptable excipients, then stored under sterilestorage conditions until shortly before use, at which time it isreconstituted with a defined volume of diluent, to bring the liquid tothe desired concentration and viscosity.

The formulations described herein may be stored by any suitable methodknown to one skilled in the art. Non-limiting examples of methods forpreparing the protein formulations for storage include freezing,lyophilizing, and spray drying the liquid protein formulation. In somecases, the lyophilized formulation is frozen for storage at subzerotemperatures, such as at about −80° C. or in liquid nitrogen. In somecases, a lyophilized or aqueous formulation is stored at 2-8° C.

Non-limiting examples of diluents useful for reconstituting alyophilized formulation prior to injection include sterile water,bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solution,dextrose solution, or aqueous solutions of salts and/or buffers. In somecases, the formulation is spray-dried and then stored.

IV. Administration to an Individual in Need Thereof

The protein formulations, including, but not limited to, reconstitutedformulations, are administered to a person in need thereof byintramuscular, intraperitoneal (i.e., into a body cavity),intracerobrospinal, or subcutaneous injection using an 18-32 gaugeneedle (optionally a thin-walled needle), in a volume of less than about5 mL, less that about 3 mL, preferably less than about 2 mL, morepreferably less than about 1 mL.

The appropriate dosage (“therapeutically effective amount”) of theprotein, such as a mAb, will depend on the condition to be treated, theseverity and course of the disease or condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered at one time in single or multiple injections,or over a series of treatments, as the sole treatment, or in conjunctionwith other drugs or therapies.

Dosage formulations are designed so that the injections cause nosignificant signs of irritation at the site of injection, for example,wherein the primary irritation index is less than 3 when evaluated usinga Draize scoring system. In an alternative embodiment, the injectionscause macroscopically similar levels of irritation when compared toinjections of equivalent volumes of saline solution. In anotherembodiment, the bioavailability of the protein is higher when comparedto the otherwise same formulation without the viscosity-loweringagent(s) administered in the same way. In another embodiment, theformulation is at least approximately as effective pharmaceutically asabout the same dose of the protein administered by intravenous infusion.

In a preferred embodiment, the formulation is injected to yieldincreased levels of the therapeutic protein. For example, the AUC valuemay be at least 10%, preferably at least 20%, larger than the same valuecomputed for the otherwise same formulation without theviscosity-lowering agent(s) administered in the same way.

The viscosity-lowering agent may also affect bioavailability. Forexample, the percent bioavailability of the protein may be at least 1.1times, preferably at least 1.2 times the percent bioavailability of theotherwise same formulation without the viscosity-lowering agent(s)administered in the same way.

The viscosity-lowering agent may also affect the pharmacokinetics. Forexample, the C_(MAX) after SC or IM injection may be at least 10%,preferably at least 20%, less than the C_(MAX) of an approximatelyequivalent pharmaceutically effective intravenously administered dose.

In some embodiments, the proteins are administered at a higher dosageand a lower frequency than the otherwise same formulations without theviscosity-lowering agent(s).

The lower viscosity formulations require less injection force. Forexample, the injection force may be at least 10%, preferably at least20%, less than the injection force for the otherwise same formulationwithout the viscosity-lowering agent(s) administered in the same way. Inone embodiment, the injection is administered with a 27 gauge needle andthe injection force is less than 30 N. The formulations can beadministered in most cases using a very small gauge needle, for example,between 27 and 31 gauge, typically 27, 29 or 31 gauge.

The viscosity-lowering agent may be used to prepare a dosage unitformulation suitable for reconstitution to make a liquid pharmaceuticalformulation for subcutaneous or intramuscular injection. The dosage unitmay contain a dry powder of one or more proteins; one or moreviscosity-lowering agents; and other excipients. The proteins arepresent in the dosage unit such that after reconstitution in apharmaceutically acceptable solvent, the resulting formulation has aprotein concentration from about 100 mg to about 2,000 mg per 1 mL(mg/mL). Such reconstituted formulations may have an absolute viscosityof from about 1 cP to about 50 cP at 25° C.

The low viscosity formulation can be provided as a solution or in adosage unit form where the protein is lyophilized in one vial, with orwithout the viscosity-lowering agent and the other excipients, and thesolvent, with or without the viscosity-lowering agent and otherexcipients, is provided in a second vial. In this embodiment, thesolvent is added to the protein shortly before or at the time ofinjection to ensure uniform mixing and dissolution.

The viscosity-lowering agent(s) are present in the formulations atconcentrations that cause no significant signs of toxicity and/or noirreversible signs of toxicity when administered via subcutaneous,intramuscular, or other types of injection. As used herein, “significantsigns of toxicity” include intoxication, lethargy, behavioralmodifications such as those that occur with damage to the centralnervous system, infertility, signs of serious cardiotoxicity such ascardiac arrhythmia, cardiomyopathy, myocardial infarctions, and cardiacor congestive heart failure, kidney failure, liver failure, difficultybreathing, and death.

In preferred embodiments the formulations cause no significantirritation when administered not more than twice daily, once daily,twice weekly, once weekly or once monthly. The protein formulations canbe administered causing no significant signs of irritation at the siteof injection, as measured by a primary irritation index of less than 3,less than 2, or less than 1 when evaluated using a Draize scoringsystem. As used herein, “significant signs of irritation” includeerythema, redness, and/or swelling at the site of injection having adiameter of greater than 10 cm, greater than 5 cm, or greater than 2.5cm, necrosis at the site of injection, exfoliative dermatitis at thesite of injection, and severe pain that prevents daily activity and/orrequires medical attention or hospitalization. In some embodiments,injections of the protein formulations cause macroscopically similarlevels of irritation when compared to injections of equivalent volumesof saline solution.

The protein formulations can exhibit increased bioavailability comparedto the otherwise same protein formulation without the viscosity-loweringagent(s) when administered via subcutaneous or intramuscular injection.“Bioavailability” refers to the extent and rate at which the bioactivespecies such as a mAb, reaches circulation or the site of action. Theoverall bioavailability can be increased for SC or IM injections ascompared to the otherwise same formulations without theviscosity-lowering agent(s). “Percent bioavailability” refers to thefraction of the administered dose of the bioactive species which enterscirculation, as determined with respect to an intravenously administereddose. One way of measuring the bioavailability is by comparing the “areaunder the curve” (AUC) in a plot of the plasma concentration as afunction of time. The AUC can be calculated, for example, using thelinear trapezoidal rule. “AUC_(∞)”, as used herein, refers to the areaunder the plasma concentration curve from time zero to a time where theplasma concentration returns to baseline levels. “AUC_(0-t)”, as usedherein, refers to the area under the plasma concentration curve fromtime zero to a time, t, later, for example to the time of reachingbaseline. The time will typically be measured in days, although hourscan also be used as will be apparent by context. For example, the AUCcan be increased by more than 10%, 20%, 30%, 40%, or 50% as compared tothe otherwise same formulation without the viscosity-lowering agent(s)and administered in the same way.

As used herein, “t_(max)” refers to the time after administration atwhich the plasma concentration reaches a maximum.

As used herein, “C_(max)” refers to the maximum plasma concentrationafter dose administration, and before administration of a subsequentdose.

As used herein, “C_(min)” or “C_(trough)” refers to the minimum plasmaconcentration after dose administration, and before administration of asubsequent dose.

The C_(max) after SC or IM injection may be less, for example, at least10%, more preferably at least 20%, less than the C_(max) of anintravenously administered dose. This reduction in C_(max) may alsoresult in decreased toxicity.

The pharmacokinetic and pharmacodynamic parameters may be approximatedacross species using approaches that are known to the skilled artisan.The pharmacokinetics and pharmacodynamics of antibody therapeutics candiffer markedly based upon the specific antibody. An approved murine mAbwas shown to have a half-life in humans of ˜1 day, while a human mAbwill typically have a half-life of 25 days (Waldmann et al., Int.Immunol., 2001, 13:1551-1559). The pharmacokinetics and pharmacodynamicsof antibody therapeutics can differ markedly based upon the route ofadministration. The time to reach maximal plasma concentration after IMor SC injection of IgG typically ranges from 2 to 8 days, althoughshorter or longer times may be encountered (Wang et al., Clin. Pharm.Ther., 2008, 84(5):548-558). The pharmacokinetics and pharmacodynamicsof antibody therapeutics can differ markedly based upon the formulation.

The low-viscosity protein formulations can allow for greater flexibilityin dosing and decreased dosing frequencies compared to those proteinformulations without the viscosity-lowering agent(s). For example, byincreasing the dosage administered per injection multiple-fold, thedosing frequency can in some embodiments be decreased from once every 2weeks to once every 6 weeks.

The protein formulations, including, but not limited to, reconstitutedformulations, can be administered using a heated and/or self-mixingsyringe or autoinjector. The protein formulations can also be pre-heatedin a separate warming unit prior to filling the syringe.

i. Heated Syringes

The heated syringe can be a standard syringe that is pre-heated using asyringe warmer. The syringe warmer will generally have one or moreopenings each capable of receiving a syringe containing the proteinformulation and a means for heating and maintaining the syringe at aspecific (typically above the ambient) temperature prior to use. Thiswill be referred to herein as a pre-heated syringe. Suitable heatedsyringe warmers include those available from Vista Dental Products andInter-Med. The warmers are capable of accommodating various sizedsyringes and heating, typically to within 1° C., to any temperature upto about 130° C. In some embodiments the syringe is pre-heated in aheating bath such as a water bather maintained at the desiredtemperature.

The heated syringe can be a self-heating syringe, i.e. capable ofheating and maintaining the liquid formulation inside the syringe at aspecific temperature. The self-heating syringe can also be a standardmedical syringe having attached thereto a heating device. Suitableheating devices capable of being attached to a syringe include syringeheaters or syringe heater tape available from Watlow ElectricManufacturing Co. of St. Louis, Mo., and syringe heater blocks, stageheaters, and in-line perfusion heaters available from Warner Instrumentsof Hamden, Conn., such as the SW-61 model syringe warmer. The heater maybe controlled through a central controller, e.g. the TC-324B or TC-344Bmodel heater controllers available from Warner Instruments.

The heated syringe maintains the liquid protein formulation at aspecified temperature or to within 1° C., within 2° C., or within 5° C.of a specified temperature. The heated syringe can maintain the proteinformulation at any temperature from room temperature up to about 80° C.,up to about 60° C., up to about 50° C., or up to about 45° C. as long asthe protein formulation is sufficiently stable at that temperature. Theheated syringe can maintain the protein formulation at a temperaturebetween 20° C. and 60° C., between 21° C. and 45° C., between 22° C. and40° C., between 25° C. and 40° C., or between 25° C. and 37° C. Bymaintaining the protein formulations at an elevated temperature duringinjection, the viscosity of the liquid formulation is decreased, thesolubility of the protein in the formulation is increased, or both.

ii. Self-Mixing Syringes

The syringe can be self-mixing or can have a mixer attached. The mixercan be a static mixer or a dynamic mixer. Examples of static mixersinclude those disclosed in U.S. Pat. Nos. 5,819,988, 6,065,645,6,394,314, 6,564,972, and 6,698,622. Examples of some dynamic mixers caninclude those disclosed in U.S. Pat. Nos. 6,443,612 and 6,457,609, aswell as U.S. Patent Application Publication No. US 2002/0190082. Thesyringe can include multiple barrels for mixing the components of theliquid protein formulation. U.S. Pat. No. 5,819,998 describes syringeswith two barrels and a mixing tip for mixing two-component viscoussubstances.

iii. Autoinjectors and Pre-Filled Syringes of Protein Formulations

The liquid protein formulation can be administered using a pre-filledsyringe autoinjector or a needleless injection device. Autoinjectorsinclude a handheld, often pen-like, cartridge holder for holdingreplaceable pre-filled cartridges and a spring based or analogousmechanism for subcutaneous or intramuscular injections of liquid drugdosages from a pre-filled cartridge. Autoinjectors are typicallydesigned for self-administration or administration by untrainedpersonnel. Autoinjectors are available to dispense either single dosagesor multiple dosages from a pre-filled cartridge. Autoinjectors enabledifferent user settings including inter alia injection depth, injectionspeed, and the like. Other injection systems can include those describedin U.S. Pat. No. 8,500,681.

The lyophilized protein formulation can be provided in pre-filled orunit-dose syringes. U.S. Pat. Nos. 3,682,174; 4,171,698; and 5,569,193describe sterile syringes containing two-chambers that can be pre-filledwith a dry formulation and a liquid that can be mixed immediately priorto injection. U.S. Pat. No. 5,779,668 describes a syringe system forlyophilization, reconstitution, and administration of a pharmaceuticalcomposition. In some embodiments the protein formulation is provided inlyophilized form in a pre-filled or unit-dose syringe, reconstituted inthe syringe prior to administration, and administered as a singlesubcutaneous or intramuscular injection. Autoinjectors for delivery ofunit-dose lyophilized drugs are described in WO 2012/010,832. Autoinjectors such as the Safe Click Lyo™ (marketed by Future InjectionTechnologies, Ltd., Oxford, U.K.) can be used to administer a unit-doseprotein formulation where the formulation is stored in lyophilized formand reconstituted just prior to administration. In some embodiments theprotein formulation is provided in unit-dose cartridges for lyophilizeddrugs (sometimes referred to as Vetter cartridges). Examples of suitablecartridges can include those described in U.S. Pat. Nos. 5,334,162 and5,454,786.

V. Methods of Purification and Concentration

The viscosity-lowering agents can also be used to assist in proteinpurification and concentration. The viscosity-lowering agent(s) andexcipients are added to the protein in an effective amount reduce theviscosity of the protein solution. For example, the viscosity-loweringagent is added to a concentration of between about 0.01 M and about 1.0M, preferably between about 0.01 M and about 0.50 M, and most preferablybetween about 0.01 M and about 0.25 M.

The viscosity-lowering agent solution containing protein is thenpurified or concentrated using a method selected from the groupconsisting of ultrafiltration/diafiltration, tangential flow filtration,centrifugal concentration, and dialysis.

EXAMPLES

The foregoing will be further understood by the following non-limitingexamples.

All viscosities of well-mixed aqueous mAb solutions were measured usingeither a mVROC microfluidic viscometer (RheoSense) or a DV2T cone andplate viscometer (Brookfield; “C & P”) after a 5 minute equilibration at25° C. (unless otherwise indicated). The mVROC viscometer was equippedwith an “A” or “B” chip, each manufactured with a 50 micron channel.Typically, 0.10 mL of protein solution was back-loaded into a gastightmicrolab instrument syringe (Hamilton; 100 μL), affixed to the chip, andmeasured at multiple flow rates, approximately 20%, 40%, and 60% of themaximum pressure for each chip. For example a sample of approximately 50cP would be measured at around 10, 20, and 30 μL/min (approximately 180,350, and 530 s⁻¹, respectively, on an “A” chip) until viscositystabilized, typically after at least 30 seconds. An average absoluteviscosity and standard deviation was then calculated from at least thesethree measurements. The C & P viscometer was equipped with a CPE40 orCPE52 spindle (cone angle of 0.8° and 3.0°, respectively) and 0.50 mLsamples were measured at multiple shear rates between 2 and 400 s⁻¹.Specifically, samples were measured for 30 seconds each at 22.58, 24.38,26.25, 28.13, 30, 31.88, 45, 67.5, 90, 112.5, 135, 157.5, 180, 202.5,247, 270, 292.5, 315, 337.5, 360, 382, 400 s⁻¹, starting at a shear ratethat gave at least 10% torque, and continuing until instrument torquereached 100%. An extrapolated zero-shear viscosity was then determinedfrom a plot of dynamic viscosity versus shear rate for the samplesmeasured on a DV2T cone and plate viscometer. The extrapolatedzero-shear viscosities reported are the average and standard deviationof at least three measurements.

Example 1: Effect of a Viscosity-Lowering Agent, Camphorsulfonic AcidLysine (CSAL), on the Viscosity of Solutions of Biosimilar ERBITUX®

Materials and Methods

A commercially-obtained biosimilar ERBITUX® (100-400 mg) containingpharmaceutical excipients (Polysorbate 80, phosphate buffer, and NaCl)was purified. First, Polysorbate 80 was removed using DETERGENT-OUT®TWEEN® Medi Columns (G-Biosciences). Next, the resulting solutions wereextensively buffer-exchanged into 20 mM sodium phosphate buffer (PB; pH7.0) or 20 mM CSAL (pH 7.0) and concentrated to a final volume of lessthan 10 mL on Jumbosep centrifugal concentrators (Pall Corp.). Thecollected protein solution was freeze-dried. The dried protein cakes,containing protein and buffer salts or agent, were reconstituted to afinal volume of 0.15-1.3 mL. These samples were reconstituted usingadditional PB (pH 7.0) or CSAL (pH 7.0) sufficient to bring the finalconcentration of PB or CSAL to 0.25 M. The final concentration of mAb insolution was determined by light absorbance at 280 nm. Reported proteinconcentrations represent the range of all protein samples included ineach Table or Figure. Specifically, reported values are the median plusor minus half the range. Extrapolated zero-shear using an experimentallydetermined extinction coefficient of 1.4 L/g·cm and viscosities reportedwere measured on a DV2T cone and plate viscometer.

Results

The data in FIGS. 1A and 1B demonstrate the viscosity-lowering effect ofCSAL on aqueous solutions of biosimilar ERBITUX®. The viscosity of asolution of biosimilar ERBITUX® in phosphate buffer (PB) increasesexponentially with increasing mAb concentration. The viscosity of asolution of biosimilar ERBITUX® in the presence of CSAL is seen toincrease exponentially with increasing mAb concentration, but to alesser extent than the formulation in PB i.e. the viscosity gradient isreduced. The data in FIGS. 1A and 1B show that the higher theconcentration of mAb, the greater the viscosity-lowering effect. Themagnitude of viscosity-lowering effects afforded by the replacement ofPB with CSAL varied from 1.1-fold at 100±5 mg/mL to 10.3-fold at 227±5mg/mL mAb.

Example 2: Viscosity-Lowering Effect of a Viscosity-Lowering Agent,Camphorsulfonic Acid Lysine (CSAL), as a Function of Concentration ofBiosimilar AVASTIN®

Materials and Methods

A biosimilar AVASTIN® obtained commercially and containingpharmaceutical excipients (Polysorbate 20, phosphate buffer, citratebuffer, mannitol, and NaCl) was purified, buffer exchanged,concentrated, dried, reconstituted, and analyzed as described in Example1 above (using the extinction coefficient of 1.7 L/g·cm at 280 nm). Theprotein was formulated to contain either 0.25 M phosphate buffer or 0.25M CSAL.

Results

FIGS. 2A and 2B depict the viscosity of aqueous mAb solutions as afunction of mAb concentration in aqueous buffered solution and withCSAL. The viscosity of biosimilar AVASTIN® in aqueous phosphate bufferand in the presence of CSAL increases exponentially with increasingconcentration; however, as in the case of biosimilar ERBITUX®, thisincrease is much less marked for the CSAL-containing formulation, i.e.the viscosity gradient is reduced. In general, the higher the mAbconcentration, the greater the viscosity-lowering effect observed. Themagnitude of viscosity-lowering effects afforded by the replacement ofPB with CSAL varied from 2.1-fold at 80 mg/mL to 3.7-fold at 230±5 mg/mLmAb.

Example 3: Viscosity-Lowering Effect as a Function of CSAL Concentrationfor Aqueous Solutions of Biosimilar ERBITUX®

Materials and Methods

Samples were purified, buffer exchanged, concentrated, dried,reconstituted, and analyzed similarly to Example 1 above. The finalconcentration of CSAL upon reconstitution in an aqueous CSAL solutionranged from 0.25 M to 0.50 M.

Results

Table 1 shows the viscosity of solutions of biosimilar ERBITUX®formulated in 0.25 M phosphate buffer (no CSAL as a control) and withvarying concentrations of CSAL. The viscosity-lowering effect of CSAL isseen to rise from 8.4- to 12.1-fold with increasing viscosity-loweringagent concentration. The data in Table 1 show that the higher theconcentration of CSAL, the greater the viscosity-lowering effect, atleast within the agent concentration range tested.

TABLE 1 Viscosities of aqueous solutions of biosimilar ERBITUX ® (155 ±5 mg/mL, pH 7.0) in the presence of different concentrations of CSAL at25° C. Fold viscosity reduction (compared to no CSAL [CSAL], MViscosity, cP present) 0 154 ± 0  1 0.25 18.3 ± 0.0 8.4 0.38 14.9 ± 0.110.3 0.50 12.7 ± 0.1 12.1

Example 4: Viscosities of Solutions of Biosimilar ERBITUX® as a Functionof Temperature in the Presence of Various Viscosity-Lowering Agents

Materials and Methods

Aqueous solutions of biosimilar ERBITUX® containing variousviscosity-lowering agents were prepared as described in Example 1.Specifically, 20 mM solutions of the viscosity-lowering agents ofinterest were used for buffer exchange, and the lyophilized cakes werereconstituted to 0.25 M of each viscosity-lowering agent. For the samplecontaining CSA-APMI, biosimilar ERBITUX® was extensively bufferexchanged into 2 mM PB (pH 7.0), and concentrated to a final volume ofless than 10 mL on Jumbosep centrifugal concentrators (Pall Corp.). Thesample was first aliquoted. Then, an appropriate amount of CSAAPMIsolution (pH 7.0) was added to each aliquot such that uponreconstitution with water, the final excipient concentration is 0.25 M.The protein solutions were then freeze-dried. The dried protein cakes,containing protein and viscosity-lowering agent (and a negligible amountof buffer salts) were reconstituted to a final volume of approximately0.10 mL and viscosity-lowering agent concentration as previouslydescribed.

Results

Table 2 shows viscosity data for biosimilar ERBITUX® in the presence ofsix viscosity-lowering agents—camphorsulfonic acid lysine (CSAL),camphorsulfonic acid arginine (CSAA), benzenesulfonic acid lysine(BSAL), benzenesulfonic acid arginine (BSAA), naphthalenesulfonic acidarginine (NSAA), and camphorsulfonic acid1-(3-aminopropyl)-2-methyl-1H-imidazole (CSAAPMI). The data in Table 2show a reduction in viscosity of at least about 9-fold for all sixviscosity-lowering agents compared to a solution of biosimilar ERBITUX®in phosphate buffer under otherwise the same conditions. The mostefficacious viscosity-lowering agent—CSAAPMI—lowered viscosity by>40-fold.

Additionally, the data in Table 3 show that at multiple temperaturesranging from 20° C. to 30° C., a 225 mg/mL solution of biosimilarERBITUX® prepared with 0.25 M CSAA had the lowest viscosity of the fiveviscosity-lowering agents. Thus, the observed trends in viscosities at25° C. seem to be predictive of those at temperatures of at least 20° C.and 30° C.

TABLE 2 Reduction in viscosity of aqueous solutions of biosimilarERBITUX ® (226 ± 6 mg/mL, pH 7.0) formulated with various 0.25Mviscosity-lowering agents, as compared to that in 0.25M sodium phosphatebuffer (PB) at 25° C. Agent Viscosity, cP Fold reduction PB 1130 ± 7  1CSAL 109 ± 1  10.4 CSAA 58.0 ± 0.3 19.5 BSAL 126 ± 1  9.0 BSAA 61.3 ±0.9 18.4 NSAA 69.4 ± 0.6 16.3 CSAAPMI 25.7 ± 1.5 44.0

TABLE 3 Viscosities of aqueous solutions of biosimilar ERBITUX ® (225 ±5 mg/mL, pH 7.0) formulated with various 0.25M viscosity-loweringagents. Viscosity, cP Agent Temp. PB CSAL CSAA BSAL BSAA NSAA 20° C.1810 ± 10 166 ± 2 79.6 ± 0.9 193 ± 0 85.2 ± 0.6 103 ± 0  25° C. 1130 ±7  109 ± 1 58.0 ± 0.3 126 ± 1 61.3 ± 0.9 69.4 ± 0.6 30° C. 723 ± 0  78.4± 1.5 46.9 ± 0.6  89.8 ± 0.8 50.5 ± 1.9 60.9 ± 4.3

Example 5: The Effect of Temperature on Viscosity of Aqueous Solutionsof Biosimilar AVASTIN® Formulated with Various Viscosity-Lowering Agents

Materials and Methods

Solutions of biosimilar AVASTIN® containing different viscosity-loweringagents were prepared as described in Example 1 above. In particular, 20mM solutions of the viscosity-lowering agents of interest were used forbuffer exchange, and the lyophilized cakes were reconstituted to 0.15 or0.25 M viscosity-lowering agent.

Results

As seen in Table 4, 0.25 M CSAL lowered the viscosity of a 230±5 mg/mLsolution of biosimilar AVASTIN® at all three temperatures between 20 and30° C. Furthermore, 0.15 M CSAL reduces viscosity to approximately thesame absolute value as 0.25 M CSAL at 20 and 25° C. and is equallyeffective at 30° C.

The data in Table 5 compare the effects of CSAL and BSAL at aconcentration of 0.15 M. CSAL is a superior viscosity-lowering agentcompared to BSAL at all three temperatures.

TABLE 4 Viscosities of aqueous solutions of biosimilar AVASTIN ® (230 ±5 mg/mL, pH 7.0) formulated with 0.25 and 0.15M CSAL at differenttemperatures. Viscosity, cP Temperature 0.25M PB 0.25M CSAL 0.15M CSAL20° C. 563 ± 2 152 ± 0 157 ± 0 25° C. 397 ± 2 107 ± 4 113 ± 0 30° C. 311± 4  95.5 ± 5.4  91.7 ± 3.3

TABLE 5 Viscosities of aqueous solutions of biosimilar AVASTIN ® (230 ±5 mg/mL, pH 7.0) formulated with 0.15M CSAL and BSAL at differenttemperatures. Viscosity, cP Temperature 0.25M PB 0.15M CSAL 0.15M BSAL20° C. 563 ± 2 157 ± 0 395 ± 3 25° C. 397 ± 2 113 ± 0 227 ± 5 30° C. 311± 4  91.7 ± 3.3 175 ± 7

Example 6: Removal of CSAL Reverses Viscosity-Lowering Effect in mAbSolutions

Materials and Methods

Three samples each of biosimilar ERBITUX® and biosimilar AVASTIN® wereprepared. First, Polysorbate was removed from the commercially obtainedmAb solutions. The resulting solution with remaining pharmaceuticalexcipients was either (i) concentrated on a centrifugal device with a100-kDa molecular weight cutoff (MWCO) (Pall Corp.) as a control sample(original excipients), (ii) buffer exchanged into 0.25 M CSAL asdescribed in Example 1, or (iii) buffer exchanged into 0.25 M CSAL asdescribed in Example 1, reconstituted, and then further exchanged into0.25 M PB. In this third instance, exchange into 0.25 M phosphate bufferproceeded first by overnight dialysis against 20 mM PB (50-kDa MWCO,Spectrum Labs). The partially dialyzed samples were then diluted to 60mL in 0.25 M PB and subjected to centrifugal concentration (30-kDa MWCOJumbosep (Pall Corp.), followed by a 100-kDa MWCO Macrosep device (PallCorp.)). The viscosities of these three aqueous solutions weredetermined as described in Example 1 above.

Results

The viscosities of aqueous solutions of both biosimilar ERBITUX® andbiosimilar AVASTIN® decreased in the presence of CSAL—2.7- and 1.5-fold,respectively—but then increased when CSAL was removed (see Tables 6 and7). Furthermore, upon removal of CSAL, mAb solution viscosities returnedto approximately the same level as the original solutions, suggestingthat CSAL does not damage the protein and showing that it is necessaryfor the observed viscosity reduction.

TABLE 6 Viscosities of aqueous solutions of biosimilar ERBITUX ® (80 ± 5mg/mL, pH 7.0) at 25° C. Formulation Viscosity, cP Original excipients8.30 ± 0.04 0.25M CSAL 3.08 ± 0.18 0.25M CSAL exchanged into 0.25M PB9.43 ± 0.04

TABLE 7 Viscosities of aqueous solutions of biosimilar AVASTIN ® (101 ±5 mg/mL, pH 7.0) at 25° C. Formulation Viscosity, cP Original excipients6.08 ± 0.19 0.25M CSAL 4.03 ± 0.24 0.25M CSAL exchanged into 0.25M PB6.61 ± 0.08

Example 7: Camphorsulfonic Acid-Containing Viscosity-Lowering AgentsProvide Large Viscosity Reductions in Aqueous Solutions of AVASTIN® andBiosimilar AVASTIN®

Materials and Methods

AVASTIN® and a biosimilar AVASTIN® obtained commercially and containingpharmaceutical excipients (AVASTIN®: trehalose, sodium phosphate buffer,and Polysorbate 20; biosimilar AVASTIN®: Polysorbate 20, phosphatebuffer, citrate buffer, mannitol, and NaCl) were purified, bufferexchanged, concentrated, freeze-dried, and reconstituted as describedabove. Samples in Table 8 were prepared as described in Example 1 above(using the protein extinction coefficient of 1.7 L/g·cm at 280 nm) andmeasured on a C & P viscometer. Viscosity-reduced samples in Table 9were prepared as described in Example 4 above, but mAb was extensivelybuffer exchanged into 2 mM PB. Subsequently, the appropriate amount ofviscosity-lowering agent was added to result in a finalviscosity-lowering agent concentration of 0.15-0.35 M uponreconstitution. Viscosities were measured using a RheoSense mVROCmicrofluidic viscometer equipped with an “A” or “B” chip.

Results

The data in Tables 8 and 9 demonstrate the viscosity-lowering effect ofdifferent viscosity-lowering agents on aqueous solutions of biosimilarAVASTIN®. Viscosity reductions up to 2.5-fold (compared to mAb solutionsin PB) are observed for aqueous solutions of biosimilar AVASTIN® in thepresence of viscosity-lowering agents containing CSA.

TABLE 8 Viscosities of aqueous solutions of biosimiar AVASTIN ® (200 ± 5mg/mL, pH 7.0) at 25° C. with various viscosity-lowering agents. Agent[Salt] (M of anion) Viscosity (cP) PB 0.25 96.8 ± 0.9 NaCl 0.25 121 ± 8 Arginine•HCl 0.25 83.2 ± 2.8 Arginine•HCl 0.3 71.8 ± 2.2 Lysine•HCl 0.25137 ± 2  BSA sodium salt 0.25 133 ± 3  CSA sodium salt 0.25 55.7 ± 0.2BSAA 0.25 75.3 ± 0.4 Benzoic acid arginine 0.15 52.2 ± 0.5 Benzoic acidarginine 0.25 51.4 ± 0.5 CSAA 0.25 48.5 ± 1.9 CSA betaine* 0.25 66.0 ±0.7 diCSA cadaverine 0.25 85.5 ± 5.2 diCSA cadaverine 0.35 65.6 ± 1.6CSA canavanine 0.15 60.5 ± 0.6 CSA canavanine 0.25 75.6 ± 3.0 CSAcarnitine* 0.25 72.4 ± 1.7 CSA dimethylpiperazine 0.25 47.4 ± 1.3 CSAdimethylpiperazine 0.35 51.7 ± 0.9 CSAL 0.25 54.9 ± 0.9Chlorotheophylline arginine 0.25 104.5 ± 6.5  Ethandisulfonatediarginine* 0.15 77.1 ± 0.3 Ethandisulfonate diarginine* 0.25 105 ± 4 MSA arginine 0.25 93.1 ± 0.9 Toluenesulfonic acid arginine 0.25 159 ± 5 Toluenesulfonic acid lysine 0.25 118 ± 1  *Contains equimolar NaCl; CSA= Camphorsulfonic acid, BSA = Benzenesulfonic acid, MSA =Methanesulfonic acid, PB = Phosphate buffer

TABLE 9 Viscosities of aqueous solutions of biosimilar AVASTIN ® (pH7.0) at 25° C. with 0.15M viscosity-lowering agents (unless otherwisenoted). [biosimilar Viscosity Agent AVASTIN] (mg/mL) (cP) 0.25M PB 220213 ± 10 0.25M PB 200 96.8 ± 0.9 CSA-piperazine 212  64.5 ± 13.1Lactobionic acid-tris 219 109 ± 5  CSA-4-aminopyridine 229 86.4 ± 1.1Glucuronic acid-tris 221 151 ± 5 

The viscosity of a 200±9 mg/mL aqueous solution of biosimilar AVASTIN®with CSAA was measured as a function of pH as depicted in FIG. 3. As pHincreases, the magnitude of the viscosity-lowering effect resulting fromthe presence of CSAA in aqueous solutions of biosimilar AVASTIN® alsoincreases, reaching a minimum viscosity and maximum viscosity-loweringeffect around pH 7. The viscosity reduction by CSAA was compared as afunction of pH for two different concentrations of biosimilar AVASTIN®.FIG. 4 demonstrates that 0.25 M CSAA results in a greater reduction inviscosity with increasing (i) concentration of the biosimilar AVASTIN®and (ii) pH.

Table 10 compares the viscosity reduction of biosimilar AVASTIN® to thatof branded AVASTIN® with and without CSAL. The branded AVASTIN® solutionhas a much higher viscosity than a solution of the biosimilar mAb in theabsence of the agent. However, the presence of 0.25 M CSAL results in a1.8- and 3.3-fold reduction in viscosity of the biosimilar and brandedAVASTIN® respectively; the viscosities of biosimilar and brandedAVASTIN® are seen to be similar in the presence of 0.25 M CSAL.

TABLE 10 Viscosities of aqueous solutions containing 205 ± 5 mg/mL ofbiosimilar AVASTIN ® or branded AVASTIN ® with or without 0.25M CSALmeasured at 25° C. and pH 7.0. Biosimilar AVASTIN ® Salt (cP) BrandedAVASTIN ® (cP) Phosphate Buffer 96.8 ± 0.9 154 ± 4  0.25M CSAL 54.9 ±0.9 46.7 ± 0.9 CSAL = camphorsulfonic acid lysine

As demonstrated in Table 11, CSA 1-(3-aminopropyl)-2-methyl-1H-imidazole(CSAAPMI) with HCl provides superior viscosity reduction than CSAL,reducing the viscosity more than 5-fold as compared to the PB controlfor a solution of 210 mg/mL biosimilar AVASTIN®.

TABLE 11 Viscosities of aqueous solutions of biosimilar AVASTIN ® withvarious viscosity-lowering agents at 25° C. and pH 7.0. Agent [Agent], M[Protein], mg/mL Viscosity, cP PB 0.25 220 213 ± 10 CSAL 0.25 210 63.0 ±1.8 CSAAPMI-2HCl 0.25 210 40.9 ± 0.5 APMI =1-(3-aminopropyl)-2-methyl-1H-imidazole

For a solution containing ˜230 mg/mL biosimilar AVASTIN®, Table 12demonstrates viscosity reduction of approximately 5-fold withsulfosalicylic acid-containing viscosity-lowering agents as well as forCSAAPMI and CSA thiamine.

TABLE 12 Viscosities of aqueous solutions containing 228 ± 5 mg/mLbiosimilar AVASTIN ® with viscosity-lowering agents at 25° C. and pH7.0. Agent Concentration Viscosity Agent [M] (cP) PB 0.25 397 ± 2  CSAA0.25 116 ± 2  CSAL 0.25 113 ± 0  Sulfosalicylic acid diarginine 0.1581.6 ± 1.7 Sulfosalicylic acid dilysine 0.25 73.4 ± 0.4 CSAAPMI-2HCl0.25 71.8 ± 3.2 CSAthiamine-2NaCl 0.15 83.7 ± 2.2 APMI =1-(3-aminopropyl)-2-methyl-1H-imidazole; CSA = camphorsulfonic acid

Example 8. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof ERBITUX® and Biosimilar ERBITUX®

Materials and Methods

Aqueous solutions of biosimilar and branded ERBITUX® containing variousviscosity-lowering agents were prepared as described in Example 1.Specifically, 20 mM solutions of the salts of interest were used forbuffer exchange, and the lyophilized cakes were reconstituted to contain0.25 M of each agent. Viscosities were measured using either a RheoSensemVROC microfluidic viscometer equipped with an “A” or “B” chip or a DV2Tcone and plate viscometer.

Results

Table 13 shows data for biosimilar ERBITUX® (222±5 mg/mL) in thepresence of five viscosity-lowering agents: CSAA, CSAL, BSAA, BSAL, andNSAA. Table 14 compares the viscosity reduction of biosimilar ERBITUX®solutions using CSAA and CSAL to arginine or lysine alone.

TABLE 13 Viscosities of aqueous solutions of biosimilar ERBITUX ® (222 ±5 mg/mL, pH 7.0) with 0.25M viscosity-lowering agents at 25° C. AgentViscosity (cP) Fold Reduction Phosphate Buffer 1130 ± 7  1.0 CSAArginine 52.5 ± 1.0 21.5 CSA Lysine 109 ± 1  10.4 BSA Arginine 53.4 ±5.5 21.2 BSA Lysine 126 ± 1  9.0 NSA Arginine 69.4 ± 0.6 16.3

TABLE 14 Viscosities of aqueous solutions of biosimilar ERBITUX ® (222 ±5 mg/mL, pH 7.0) with 0.25M viscosity-lowering agents at 25° C. AgentViscosity (cP) Fold Reduction Phosphate Buffer 1130 ± 7  1.0 CSAA 52.5 ±1.0 21.5 CSA Sodium 393 ± 14 2.9 Arginine HCl 45.3 ± 0.5 24.9 CSAL 109 ±1  10.4 Lysine HCl 128 ± 2  8.8

The data in Table 13 show a reduction in viscosity of at least 9.0-foldfor all five viscosity-lowering agents compared to an aqueous solutionof biosimilar ERBITUX® in phosphate buffer under otherwise the sameconditions. The most efficacious viscosity-lowering agents, CSAA andBSAA, lowered the solution viscosity some 21-fold. The viscosities ofaqueous solutions of biosimilar ERBITUX® containing 0.25 M CSAA werecompared as a function of pH at varying protein concentrations. FIG. 5demonstrates that a viscosity minimum is observed around pH 7.0 for allprotein concentrations. The effect of pH on viscosity is most pronouncedfor higher protein concentrations (253 mg/mL in the example).

As seen in Table 15, the aqueous solutions of biosimilar and brandedERBITUX® have similar viscosities in the presence of the arginine saltBSAA at 0.25 M.

TABLE 15 Viscosities of 224 ± 4 mg/mL aqueous solutions of biosimilarERBITUX ® or branded ERBITUX ® with or without 0.25M BSAA at 25° C. andpH 7.0. Biosimilar ERBITUX ® Branded ERBITUX ® Agent viscosity (cP)viscosity (cP) Phosphate Buffer 1130 ± 7  556 ± 20 0.25M BSAA 53.4 ± 5.544.1 ± 0.5

The impact of the viscosity-lowering agents on the formation ofirreversible protein aggregates was examined for biosimilar ERBITUX®.Aqueous liquid formulations were prepared of (i) biosimilar ERBITUX® and(ii) biosimilar ERBITUX® containing 0.25 M CSAL. These solutions werestored for 90 days at 4° C. and pH 5.4 and 7.0, respectively. The storedsamples were examined using size exclusion chromatography (column: TosohTSKgel UltraSW Aggregate; mobile phase: 0.1 M potassium phosphate/0.1 Msodium sulfate, pH 6.8 at 0.8 mL/min; injection: 20 μL of a 5 mg/mL mAbsolution). The data in Table 16 reveal no significant aggregateformation in either the commercial drug product or high-concentrationviscosity-lowered formulation.

TABLE 16 Percentage of protein aggregate formation after 90 days ofstorage at 4° C. as measured by size exclusion chromatography foraqueous solutions containing of biosimilar ERBITUX ® with or without0.25M CSAL. Sample % Monomer % Dimer % Aggregate Biosimilar ERBITUX ®99.0 1.0 0.0 5 mg/mL Biosimilar ERBITUX ® 98.4 0.9 0.7 210 mg/mL with0.25M CSAL

Example 9. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof REMICADE®

Materials and Methods

Commercially-obtained REMICADE® containing pharmaceutical excipients(sucrose, Polysorbate 80, sodium phosphate buffer) was prepared as perinstructions in the prescribing information sheet. Subsequently, theaqueous drug product was purified, buffer exchanged, concentrated,dried, reconstituted, and analyzed as described in Example 1 above(using the extinction coefficient of 1.4 L/g*cm at 280 nm). Viscositieswere measured using either a RheoSense mVROC microfluidic viscometerequipped with an “A” or “B” chip.

Results

The data for aqueous REMICADE® solutions in Table 17 demonstrate that(i) viscosity-lowering agents containing a bulky cyclic group providegreater than 15-fold viscosity reductions, and (ii) CSAA, CSAAPMI, andsulfosalicylic acid diarginine (SSA DiArg) provide the greatestviscosity reduction of about 29-fold. Solution viscosities in thepresence of ArgHCl alone are significantly higher than those with thebulky cyclic groups.

TABLE 17 Viscosities of aqueous solutions of REMICADE ® containing 0.25Mviscosity-lowering agents at 25° C. and pH 7.0. [REMICADE ®] Viscosity(cP) (mg/mL) PB ArgHCl CSAA CSA APMI BSAA CSAL SSA DiArg 222 ± 6 1557 ±22 486 ± 34 53.7 ± 9.3 56.3 ± 2.7 92.3 ± 1.4 95.3 ± 1.1 55.9 ± 1.8 166 ±4  513 ± 15 110 ± 1  19.1 ± 0.2 31.7 ± 0.3 26.7 ± 1.2 27.4 ± 0.2 27.1 ±0.3 PB = phosphate buffer; ArgHCl = arginine HCl; CSAA = camphorsulfonicacid arginine; CSA APMI = camphorsulfonic acid1-(3-aminopropyl)-2-methyl-1H-imidazole; BSAA = benzene sulfonic acidarginine; CSAL = camphorsulfonic acid lysine; SSA DiArg = sulfosalicylicacid di-arginine.

The dependence of the viscosity reduction on the agent concentration wasexamined for aqueous solutions of REMICADE® in the presence of CSAA. Theresults presented in Table 18 demonstrate that viscosity reductionincreases with increasing agent concentration. The viscosity reduction,for example, is more than twice as large (the viscosity is less thanhalf) with 0.35 M agent as compared to 0.20 M agent.

TABLE 18 Viscosity of an aqueous solution of REMICADE ® (215 ± 5 mg/mL)in the presence of various concentrations of CSAA measured at 25° C. andpH 7.0. [CSAA], (M) Viscosity (cP) 0 1557 ± 22  0.20 81.3 ± 1.0 0.2553.7 ± 9.3 0.35 38.2 ± 0.9

Biophysical properties of solutions of REMICADE® formulated with 0.25 MCSAA were assessed over 90 days. Samples of REMICADE® formulated with0.25 M CSAA were prepared as described in Example 1 above. As seen inTable 19 and FIGS. 6A and 6B, the monomer content of concentratedsolutions of REMICADE® in 0.25 M CSAA as determined by size exclusionchromatography (Tosoh TSKgel UltraSW Aggregate column; 0.1 M potassiumphosphate/0.1 M sodium sulfate buffer pH 6.8 at 0.8 mL/min; 20 μLinjection of ˜4.5 mg/mL solutions), is similar to the drug product atall time points and no detectable aggregation is observed after storagefor 100 days at 4° C. The viscosity, as measured using a microfluidicviscometer, was demonstrated to remain stable after storage for 30 daysat 4° C. (Table 20). Additionally, antigen binding of this processedREMICADE® protein was measured with a REMICADE®-specific ELISA assay andno decrease in binding was seen between days 0 and 100 (Table 20).Similarly, the monomer content (Table 21) and antigen binding(normalized to that of the drug product, Table 22) of concentratedsolutions of REMICADE® in 0.25 M CSAA are comparable to the drug productafter 1 week of storage at room temperature. Lastly, Table 23demonstrates that storage of a lyophilized cake containing CSAA at 4° C.for 75 days has no negative effects on the solution viscosity or extentof protein aggregation when the sample is reconstituted. The results inTables 19-23 and FIGS. 6A and 6B demonstrate the biophysical stabilityof REMICADE® formulated with CSAA before and after storage for at least100 days at 4° C.

TABLE 19 No increased aggregation (compared to drug product) is observedin an aqueous solution of REMICADE ® (227 mg/mL, pH 7) after formulationwith 0.25M CSAA and storage at 4° C. Day % monomer Drug Product 99.9 ±0.03  0 99.7 ± 0.07  30 99.7 ± 0.04 100 99.9 ± 0.1 

TABLE 20 Reduced viscosity and antigen binding are retained over time inan aqueous solution of REMICADE ® (227 mg/mL, pH 7) after formulationwith 0.25M CSAA and storage at 4° C. Viscosity % binding Day (cP)(ELISA) 0 65.2 ± 0.7 105 ± 14 30 62.2 ± 1.4  98 ± 12 100 n.d. 101 ± 5 

TABLE 21 No increased aggregation (compared to drug product) is observedin an aqueous solution of REMICADE ® (219 mg/mL, pH 7) after formulationwith 0.25M CSAA and storage at room temperature. % monomer 0.25M DayDrug Product CSAA 0 99.7 ± 0.1 99.9 ± 0.1 4 99.9 ± 0.1 97.9 ± 0   7 100± 0  100 ± 0 

TABLE 22 Antigen binding persists in an aqueous solution of REMICADE ®(219 mg/mL, pH 7) after formulation with 0.25M CSAA and storage at roomtemperature. % binding (normalized to drug product) 0.25M Day DrugProduct CSAA 0 100 ± 12 88.6 ± 5.2 7 100 ± 28 114 ± 2.4

TABLE 23 REMICADE ® stored as a lyophilized powder retains low viscosityand monomer content upon reconstitution after storage at 4° C. for 75days Storage time (days) Viscosity, cP % Monomer (SEC) 0 65.2 ± 0.7 99.7± 0.1 75 59.3 ± 1.0 98.9 ± 0.1

Example 10. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof HERCEPTIN®

Materials and Methods

Commercially-obtained HERCEPTIN® containing pharmaceutical excipients(histidine buffer, trehalose, Polysorbate 20) was prepared as perinstructions in the prescribing information sheet. Subsequently, theaqueous drug product was purified, buffer exchanged, concentrated,dried, reconstituted, and analyzed as described in Example 1 above(using the extinction coefficient of 1.5 L/g*cm at 280 nm). Viscositieswere measured using a RheoSense mVROC microfluidic viscometer equippedwith an “A” or “B” chip.

Results

The data presented in Table 24 show that the viscosity of an aqueoussolution of HERCEPTIN® containing viscosity-lowering agents—compared tothose containing PB—is lowest in the presence of CSAA. At higher proteinconcentrations (i.e. >250 mg/mL) Arginine HCl alone reduces viscositysignificantly and CSA further enhances the effect.

TABLE 24 Viscosities of aqueous solutions of HERCEPTIN ® containing0.25M salts at 25° C. and pH 7.0. [HERCEPTIN ®] Viscosity (cP) (mg/mL)PB ArgHCl CSAA BSAA 270 ± 6 400 ± 4 179 ± 17 96.7 ± 4.7 115 ± 6  254 ± 3172 ± 5 116 ± 24 78.0 ± 8.7 75.4 ± 5.0 216 ± 0 n.d. 44.8 ± 1.1 55.7 ±2.3 n.d. PB = phosphate buffer; ArgHCl = arginine HCl; n.d. = notdetermined

Example 11. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof TYSABRI®

Materials and Methods

Commercially-obtained TYSABRI® containing pharmaceutical excipients(sodium phosphate buffer, sodium chloride, Polysorbate 80) was purified,buffer exchanged, concentrated, dried, reconstituted, and analyzed asdescribed in Example 1 above (using the extinction coefficient of 1.5L/g*cm at 280 nm). Viscosities were measured using a RheoSense mVROCmicrofluidic viscometer equipped with an “A” or “B” chip.

Results

The data presented in Table 25 show that the viscosity reduction of anaqueous solution of TYSABRI® containing viscosity-lowering agents isapproximately 2.5-fold (compared to solution containing PB) near 276mg/mL protein.

TABLE 25 Viscosities of aqueous solutions of TYSABRI ® containing 0.25Mviscosity-lowering agents at 25° C. and pH 7.0. [TYSABRI ®] Viscosity(cP) (mg/mL) PB ArgHCl CSAA BSAA 276 ± 8 255 ± 5 97.2 ± 5.7 92.9 ± 2.6n.d. 237 ± 4 182 ± 6 52.3 ± 4.5 47.1 ± 2.1 n.d. 230 ± 2 n.d. 37.0 ± 0.1n.d. 34.9 ± 1.3 PB = phosphate buffer; ArgHCl = arginine HCl; n.d. = notdetermined.

Example 12. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof Biosimilar RITUXAN®

Materials and Methods

Commercially-obtained biosimilar RITUXAN® containing pharmaceuticalexcipients (citrate buffer, sodium chloride, and TWEEN® 80) waspurified, buffer exchanged, concentrated, dried, reconstituted, andanalyzed as described in Example 1 above (using the extinctioncoefficient of 1.7 L/g*cm at 280 nm). Viscosities were measured using aRheoSense mVROC microfluidic viscometer equipped with an “A” or “B”chip.

Results

The data presented in Table 26 show that the viscosity reduction for anaqueous solution of biosimilar RITUXAN® containing viscosity-loweringagents is over 13-fold at approximately 213 mg/mL protein and over5-fold at approximately 202 mg/mL, compared to the mAb formulated in PB.

TABLE 26 Viscosities of aqueous solutions of biosimilar RITUXAN ® withviscosity-lowering agents at 25° C. and pH 7.0. Arg Arg SSA SSA CSA CSACSA [RITUXAN ®] PB HCl HCl diArg diAPMI Na CSAA APMI DMF (mg/mL) 0.25M0.25M 0.45M 0.25M 0.25M 0.25M 0.25M 0.25M 0.25M 213 ± 4 636 ± 32 99.9 ±5.0 86.8 ± 1.8* 68.3 ± 0.8* 46.6 ± 1.9 211 ± 2  103 ± 0  78.6 ± 2.0 161± 4 202 ± 2 251 ± 1  n.d. 46.9 ± 0.8  44.1 ± 0.1  n.d. 76.1 ± 1.3 78.4 ±0.3 38.7 ± 0.7 n.d. *[RITUXAN ®] is 220 mg/mL DMP = dimethylpiperazine

Example 13. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof VECTIBIX®

Materials and Methods

Commercially-obtained VECTIBIX® containing pharmaceutical excipients waspurified, buffer exchanged, concentrated, dried, reconstituted, andanalyzed as described in Example 1 above (using the extinctioncoefficient of 1.25 L/g*cm at 280 nm). Viscosities were measured using aRheoSense mVROC microfluidic viscometer equipped with an “A” or “B”chip.

Results

The data presented in Table 27 show that the viscosity reduction of anaqueous solution of VECTIBIX® containing viscosity-lowering agents isapproximately 2-fold at 291 mg/mL and 3-fold at 252 mg/mL, compared tosolutions with PB but no viscosity-lowering agents.

TABLE 27 Viscosities of aqueous solutions of VECTIBIX ® with 0.25Mviscosity-lowering agents at 25° C. and pH 7.0. [VECTIBIX ®] Viscosity(cP) (mg/mL) PB ArgHCl CSAA 291 ± 3 328 ± 12 n.d. 162 ± 1  264 n.d n.d.44.3 ± 2.3 252 ± 3 80.3 ± 3.3 36.2 ± 1.0 27.4 ± 1.2 233 ± 4 38.7 ± 1.824.7 ± 1.3 26.2 ± 6.5

Example 14. The Effect of Viscosity-Lowering Agents on Aqueous Solutionsof ARZERRA®

Materials and Methods

Commercially-obtained ARZERRA® containing pharmaceutical excipients waspurified, buffer exchanged, concentrated, dried, reconstituted, andanalyzed as described in Example 1 above (using the extinctioncoefficient of 1.5 L/g*cm at 280 nm). Viscosities were measured using aRheoSense mVROC microfluidic viscometer equipped with an “A” or “B”chip.

Results

The data presented in Table 28 show that the viscosity reduction of anaqueous solution of ARZERRA® containing viscosity-lowering agents isapproximately 3-fold at 274 mg/mL and 2-fold at 245 mg/mL, compared tosolutions with PB but no viscosity-lowering agents.

TABLE 28 Viscosities of aqueous solutions of ARZERRA ® with 0.25Mviscosity-lowering agents at 25° C. and pH 7.0. [ARZERRA ®] Viscosity(cP) (mg/mL) PB CSAA CSAAPMI 274 ± 10 349 ± 2 125 ± 7 98.9 ± 0.7 245 ±4  120 ± 4 n.d. 53.6 ± 0.6

Example 15. Comparison of Different Methods for Measuring Viscosity

Materials and Methods

Aqueous solutions containing 220 mg/mL REMICADE® and 0.25 M CSAA wereprepared as described above Example 1. The viscosities at 25° C. and pH7.0 are reported in Table 29 as extrapolated zero-shear viscosities fromcone and plate viscometer measurements and as absolute viscositiesmeasured with a microfluidic viscometer. The cone and plate measurementsused a DV2T cone and plate viscometer (Brookfield) equipped with a CPE40or CPE52 spindle measured at multiple shear rates between 2 and 400 s⁻¹.An extrapolated zero-shear viscosity was determined from a plot ofabsolute viscosity versus shear rate. The microfluidic viscometermeasurements were performed using a RheoSense mVROC microfluidicviscometer equipped with an “A” or “B” chip at multiple flow rates(approximately 20%, 40%, and 60% of the maximum pressure for each chip).

Results

The data in Table 29 demonstrates that the absolute viscosities from themicrofluidic viscometer can be directly compared to the extrapolatedzero-shear viscosities determined from the cone and plate viscometer.

TABLE 29 Viscosities of aqueous solutions of REMICADE ® (220 mg/mL) with0.25M CSAA at 25° C. and pH 7.0 measured on two different viscometers.Instrument Viscosity (cP) Cone and plate 62.3 ± 0.1 viscometer (C&P)Microfluidic viscometer 53.7 ± 9.3 on a chip (mVROC)

In order to compare a broader range of viscosities and proteinconcentrations, aqueous solutions of a model antibody, bovine gammaglobulin, were prepared with and without 0.25 M CSAL. The viscositieswere measured as described above at protein concentrations ranging from110 mg/mL to 310 mg/mL. The data presented in Table 30 demonstrates thatthe absolute viscosities from the microfluidic viscometer can bedirectly compared to the extrapolated zero-shear viscosities for bothlow and high viscosity protein solutions.

TABLE 30 Viscosities of aqueous gamma globulin solutions with andwithout 0.25M CSAL at 25° C. and pH 7.0 measured on two differentviscometers. [gamma Viscosity (cP) globulin] without CSAL With CSAL(mg/mL) C & P microfluidic C & P microfluidic 110  3.81 ± 0.19  2.66 ±0.01 n.d. n.d. 170  12.0 ± 0.6  11.0 ± 0.1 10.3 ± 1.0 10.6 ± 0.1 260 167± 1 161 ± 1 93.5 ± 1.2 85.3 ± 0.3 310 399 ± 1 377 ± 2 223 ± 1  203 ± 2 

Example 16. Viscosity-Lowering Agents Show No Signs of Toxicity whenInjected Subcutaneously

Materials and Methods

Thirty 11-week old Sprague-Dawley rats were separated into 6 groups of 5rats each (3 saline control groups and 3 CSAA groups). The rats wereinjected subcutaneously with 0.5 mL of either endotoxin-freephosphate-buffered saline or endotoxin-free 0.25 M CSAA according to thefollowing schedule: One group from each condition was injected once onday 1 and then sacrificed 1 hour later; one group from each conditionwas injected once on day 1 and once on day 2 and then sacrificed 24hours after the second injection; and one group from each condition wasinjected once on day 1, once on day 2, and once on day 3, and thensacrificed 24 hours after the third injection.

Clinical observations were recorded for any pharmaco-toxicological signspre-dose, immediately post-dose, at 1 and 4 hours (±15 minutes)post-dose, and daily thereafter. Irritation, if any, at injection siteswas scored using the Draize evaluation scores pre-dose, immediatelypost-dose, at 1 hour (±15 minutes) post dose, and prior to sacrifice.

Results

Overall, the observed consequences of the injections of saline and CSAAwere macroscopically similar throughout the course of the study. Bothinduced from no irritation to slight irritation with edema scores of 0-2at various time points. Microscopic examination of injection sitessuggests a very minor, clinically insignificant, irritative effect withCSAA that was no longer evident by day 4.

Example 17. Concentrated Aqueous Solutions of REMICADE® Formulated withViscosity-Lowering Agents Exhibit Low Syringe Extrusion Forces and HighMonomer Content when Expelled Through Various Gauge Needles

Materials and Methods

Commercially-obtained REMICADE® containing pharmaceutical excipients(sucrose, Polysorbate 80, sodium phosphate buffer) was prepared perinstructions in the prescribing information sheet. Subsequently, theaqueous drug product was purified, buffer exchanged, concentrated,dried, reconstituted, and analyzed as described in Example 1 above(using the extinction coefficient of 1.4 L/g*cm at 280 nm). 20 mMsolutions of either phosphate buffer, CSAAPMI or CSAA were used forbuffer exchange, and the lyophilized cakes were reconstituted to 0.25 Mof each viscosity-lowering agent. Following reconstitution, theviscosity of each solution was measured using the microfluidicviscometer as described in previous examples. The solutions were thenback-loaded into 1 mL BD insulin syringes with 27, 29, or 31 gauge fixedneedles. The force required to extrude the concentrated REMICADE®solutions was then measured using an Instron at a rate of displacementequivalent to a fluid flow rate of 3 mL/min. The expelled solution wascollected from the syringe and analyzed by size-exclusionchromatography.

Results

All REMICADE® solutions containing viscosity-lowering agents were ableto be expelled through the syringes at relatively low extrusion forces(Table 31). The solution containing phosphate buffer could not beexpelled due to high viscosity. Both solutions containingviscosity-lowering agents retained high monomer content post-extrusionregardless of needle gauge, as indicated in Table 31.

TABLE 31 Syringeability of concentrated aqueous solutions of REMICADE ®extruded through various gauge needles. [REMICADE ®] (mg/mL) NeedleSyringe Agent (viscosity in cP) gauge % Monomer Force (N) 0.25M 220(1,500) pre-syringe 98.8 ± 0.0 na Phosphate 27 could not be na Buffer 29extruded 31 0.25M 230 (90.8 ± 8.4) pre-syringe  99.2 ± 0.32 na CSAAPMI27 99.1 ± 0.0 21.9 29 99.0 ± 0.0 30.4 31 99.0 ± 0.0 38.4 0.25M 224 (60.9± 1.1) pre-syringe 99.7 ± 0.3 na CSAA 27 99.5 ± 0.1 18.4 29 99.4 ± 0.224.9 31 99.5 ± 0.2 33.0

Example 18: Viscosity-Lowering Agents Reduce the Viscosity ofConcentrated Aqueous Solutions of Biosimilar AVASTIN®

Materials and Methods

A commercially-obtained biosimilar AVASTIN® containing pharmaceuticalexcipients (Polysorbate 20, phosphate and citrate buffers, mannitol, andNaCl) was purified. First, Polysorbate 20 was removed usingDETERGENT-OUT® TWEEN Medi Columns (G-Biosciences). Next, the resultingsolutions were extensively buffer-exchanged into 20 mM sodium phosphatebuffer (PB) for PB samples and 2 mM PB for viscosity-lowering agentsamples, and concentrated to a final volume of less than 10 mL onJumbosep centrifugal concentrators (Pall Corp.). The viscosity-loweringagent was then added to the 2 mM PB samples as described in Example 4above. The viscosity-lowering agent(s) were added in an amountsufficient to give concentration upon reconstitution as specified below.In cases of combinations of agents, the concentration of each componentis 0.15 M. The protein solutions were then freeze-dried. The driedprotein cakes were reconstituted in phosphate buffer (for PB samples) orwater (for samples containing viscosity-lowering agents) to a finalvolume of approximately 0.10 mL. The final concentration of mAb insolution was determined by either a Coomassie protein quantificationassay by comparing unknown concentrations of samples to a standard curveof biosimilar AVASTIN® or by A280 using the extinction coefficient of1.7 L/g*cm, when possible. Viscosities reported were measured on aRheoSense mVROC microfluidic viscometer. Results are reported in Table32.

Results

Many GRAS, IIG, and API compounds are capable of reducing the viscosityof concentrated biosimilar AVASTIN® solutions relative tophosphate-buffered samples. Of those compounds included in Table 32,local anesthetics such as procaine and lidocaine, as well as GRAS agentssuch as biotin are among the most efficacious viscosity reducingexcipients.

TABLE 32 Effect of Viscosity-Lowering Agents on Solutions of BiosimilarAVASTIN ®. [Biosimilar AVASTIN ®], Agent mg/ml Viscosity, cP 0.25MPhosphate Buffer 235 397 ± 2  220 213 ± 10 200 96.8 ± 0.9CSA-1-o-tolybiguanide 228 121 ± 1  HEPES-Tris 214 90.5 ± 1.8CSA-Na-Creatinine 202 38.4 ± 0.9 CSA-Na-aminocyclohexane carboxylic 18251.4 ± 0.1 acid 225 69.2 ± 3.7 Ethane disulfonate-diTris-2Na 219 >150CSA-piperazine† 212 ± 0  64.5 ± 13.1 Sulfacetamide-Na 214 113 ± 1 Trimetaphosphate-3Na 211 121 ± 6  CSA-Tris 206 64.4 ± 1.4 197 50 ± 1Creatinine (0.6M) 243 50.8 ± 0.5 Creatinine (0.3M) 192 24.5 ± 0.7Creatinine 232 72.7 ± 0.8 218 53.4 ± 1.0 194 36.1 ± 0.2 Lactobionicacid-Tris 219 109 ± 5  CSA-4-amino pyridine 229 86.4 ± 1.1 Sucralose 230147 ± 4  Quaternium 15 232 172 ± 4  Glucuronic acid-Tris 221 151 ± 5.0Biotin-Na 189 45.1 ± 0.9 213 60.7 ± 0.6 Procaine HCl 188 40.8 ± 0.9 22265.8 ± 0.8 Lidocaine HCl 237 97.3 ± 1.8 N-(4-Pyridiyl)pyridinium Cl HCl221 68.5 ± 1.1 Creatinine Thiamine HCl 228 59.6 ± 0.5 Pyridoxine 227 107± 0  Riboflavin-5-phosphate 225 131 ± 4  CSA Triethanolamine 238 144 ±1  Lidocaine HCl 218 147 ± 15 Chloroquine Phosphate (0.10M) 200 27.9 ±0.6 219 58.6 ± 1.6 228 71.8 ± 0.9 Scopolamine HBr 210 35.3 ± 1.1 22364.0 ± 0.8 238 87.8 ± 1.5 Levetiracetam 195 31.8 ± 0.3 192 37.1 ± 1.3215 85.5 ± 3.7 Cimetidine HCl 203 53.8 ± 2.4 Metoclopramide HCl 230 64.4± 1.6 Sumatriptan Succinate (0.25M) 212 93.2 ± 2.7 Phenylephrine HCl 201108 ± 1  Cidofovir hydrate (0.02M) 210 121 ± 2  Mepivacaine HCl 223 129± 3  Clindamycin Phosphate 200 164 ± 17 Piperacillin sodium salt 206 197± 5  Colistin sulfate salt 240 261 ± 58 Ceftriaxone sodium salt 198 301± 5  Cefazolin 229 60.6 ± 1   Granisetron HCl 168 37.9 ± 0.6 237 308 ±34 †Average of two biological replicates CSA = camphorsulfonic acid.

Example 19. Viscosity Reduction is an Agent-Concentration-DependentEffect

Materials and Methods

Aqueous solutions of a commercially-obtained biosimilar AVASTIN® wereprepared as described in Example 4. The dried protein cakes werereconstituted in phosphate buffer or water to a final volume of about0.10 mL and a final 1-(3-aminopropyl)-2-methyl-1H-imidazoledihydrochloride (APMI*2HCl) concentration of either 0.10 or 0.25 M. Thefinal concentration of mAb in solution was determined by a Coomassieprotein quantification assay by comparing unknown concentrations ofsamples to a standard curve of biosimilar AVASTIN®. Viscosities reportedwere measured on a RheoSense mVROC microfluidic viscometer.

Results

As depicted in FIG. 7, viscosity-lowering effect was increased as theconcentration of APMI*2HCl was increased.

Example 20. A Single Viscosity-Lowering Agent Lowers the Viscosity ofMany Therapeutically Relevant Monoclonal Antibodies

Materials and Methods

Aqueous solutions of a commercially-obtained biosimilar AVASTIN® wereprepared as described in Example 4. The dried protein cakes werereconstituted in phosphate buffer or water to a final volume of about0.10 mL and a final thiamine HCl concentration of 0.10 or 0.25 M. Thefinal concentration of mAb in solution was determined by a Coomassieprotein quantification assay by comparing unknown concentrations ofsamples to a standard curve of biosimilar AVASTIN®.

Commercially-obtained TYSABRI® containing pharmaceutical excipients(sodium phosphate buffer, NaCl, Polysorbate 80) was purified, bufferexchanged, concentrated, dried, reconstituted, and analyzed in the samemanner. Commercially-obtained HERCEPTIN® containing pharmaceuticalexcipients (sodium phosphate buffer, NaCl, Polysorbate 80) was purified,buffer exchanged, concentrated, dried, reconstituted, and analyzed inthe same manner. Commercially obtained biosimilar ERBITUX® containingpharmaceutical excipients (Polysorbate 80, phosphate buffer, and NaCl)was purified, buffer exchanged, concentrated, dried, reconstituted andanalyzed in the same manner. Commercially-obtained REMICADE® containingpharmaceutical excipients (sucrose, Polysorbate 80, sodium phosphatebuffer) was prepared as per instructions in the prescribing informationsheet. Subsequently, the aqueous drug product was purified, bufferexchanged, concentrated, dried, reconstituted, and analyzed as describedin the same manner. Viscosities reported were measured on a RheoSensemVROC microfluidic viscometer.

Results

The data in Table 33 demonstrate that thiamine HCl can lower theviscosity of concentrated aqueous solutions of many therapeuticallyrelevant mAbs. Thiamine HCl can produce a greater than 4-fold viscosityreduction for each mAb.

TABLE 33 Effect of Thiamine HCl on Solution Viscosity. [Protein], mAbAgent [Excipient], M mg/mL Viscosity, cP Biosimilar PB 0.25 220 213 ± 10AVASTIN ® 195 96.8 ± 0.9 Thiamine 0.25 225 53.3 ± 6.8 HCl 0.1 190 31.5 ±1.7 TYSABRI ® PB 0.25 237 182 ± 6  Thiamine 0.1 244 43.4 ± 0.7 HClHERCEPTIN ® PB 0.25 253 172 ± 4  Thiamine 0.1 218 41.6 ± 0.5 HClBiosimilar PB 0.25 235 1370 ± 3  ERBITUX ® Thiamine 0.15 245 29.5 ± 0.9HCl REMICADE ® PB 0.25 176 432 ± 30 Thiamine 0.15 178 40.7 ± 0.3 HCl

Examples 21-24. Viscosity-Lowering Agents Reduce the Viscosity ofAqueous Solutions of Many Therapeutically Relevant Monoclonal Antibodies

Materials and Methods

Aqueous solutions of commercially-obtained biosimilar RITUXAN®,TYSABRI®, HERCEPTIN®, biosimilar ERBITUX®, and REMICADE® were preparedas described in Examples 18 and 19. Tables 34-38 demonstrate thatviscosity-lowering agents can be advantageously employed for manydifferent monoclonal antibodies.

Results

TABLE 34 Viscosities of Aqueous Solutions of Biosimilar RITUXAN ®in thePresence of 0.15M Viscosity-Lowering Agents [biosimilar RITUXAN ®],Agent mg/ml Viscosity, cP 0.25M Phosphate Buffer 240 1270 ± 153 215 636± 32 199 251 ± 1  CSA-1-o-tolybiguanide 190 40.4 ± 1.9 HEPES-Tris 19150.0 ± 3.8 CSA-Na-Creatinine (0.3M) 190 33.3 ± 1.1CSA-Na-aminocyclohexane carboxylic 191 61.3 ± 2.5 acid Ethanedisulfonate-diTris-2Na 191  80.3 ± 16.0 CSA-piperazine 191 57.5 ± 0.4Sulfacetamide-Na 181 64.1 ± 1.6 Trimetaphosphate-3Na 199 126 ± 3.3CSA-Tris 191 59.1 ± 0.7 Creatinine (0.6M) 197 28.4 ± 0.2 Creatinine 20371.8 ± 0.8 Lactobionic acid-Tris 211 130 ± 1  CSA-4-amino pyridine 23366.5 ± 0.8 195 47.0 ± 1.4 Sucralose 234 111 ± 8  Quaternium 15 221 135 ±5  Glucuronic acid-Tris 207 149 ± 13 CSA-Na-Ornidazole 242 63.0 ± 3.5188 40.7 ± 0.5 Biotin-Na† 191 ± 3  96.8 ± 12.2 Procaine HCl 222 46.2 ±1.1 195 33.4 ± 1.2 Metoclopramide HCl 194 39.3 ± 0.4 Scopolamine HBr 19742.3 ± 1.0 Mepivacaine HCl 185 46.8 ± 0.6 Cimetidine HCl 215 49.5 ± 1.2Granisetron HCl 204 51.2 ± 0.8 Phenylephrine HCl 193 57.1 ± 2.8Chloroquine Phosphate (0.10M) 210 67.1 ± 1.1 Penicillin G sodium salt207 114 ± 7  Piperacillin sodium salt 194 127 ± 2  Levetiracetam 205 130± 2  Moxifloxacin HCl 193 152 ± 8  Ceftriaxone sodium salt 222 198 ± 17Clindamycin Phosphate 203 199 ± 8  Colistin sulfate salt 230 228 ± 19Cefazolin 206 65.1 ± 1.8 †Average of two biological replicates

TABLE 35 Viscosities of Aqueous Solutions of TYSABRI ® in the Presenceof 0.15M Viscosity-Lowering Agents (Unless Otherwise Indicated).[TYSABRI ®], Agent mg/mL Viscosity, cP PB 310  715 ± 106 278 255 ± 5 237 182 ± 6  Creatinine (0.30M) 219 40.8 ± 1.8 Procaine HCl 228 45.1 ±1.5 Biotin Na 233 75.8 ± 0.4 Thiamine HCl (0.10M) 244 43.4 ± 0.7

TABLE 36 Viscosities of Aqueous Solutions of HERCEPTIN ® in thePresenceof 0.15M Viscosity-Lowering Agents (Unless Otherwise Indicated).[HERCEPTIN ®], Viscosity, Agent mg/mL cP PB 272 400 ± 4  253 172 ± 5 239 122 ± 17 218 71.6 ± 3.9 Creatinine (0.3M) 222 45.7 ± 0.3 ProcaineHCl 222 41.8 ± 0.6 CSA piperazine 236 50.3 ± 0.6 CSA-Na Ornidazole 23260.1 ± 0.6 Biotin-Na 230 69.9 ± 2.3 Thiamine HCl (0.10M) 245 41.5 ± 0.5

TABLE 37 Viscosities of Aqueous Solutions of ERBITUX ® in the Presenceof 0.15M Viscosity-Lowering Agents (Unless Otherwise Indicated).[ERBITUX ®], Agent mg/mL Viscosity, cP PB 235 1370 ± 3  228 1130 ± 7 Creatinine (0.30M) 240 131 ± 4  Procaine HCl 230 35.9 ± 0.3 LidocaineHCl 223 33.8 ± 0.4 Nicotinamide 232 292 ± 10 Riboflavin-5-Phosphate(0.10M) 237 492 ± 9  Cimetidine HCl 183 19.7 ± 0.2 Metoclopramide HCl172 23.0 ± 0.2 Granisetron HCl 180 23.0 ± 0.2 Scopolamine HBr 173 23.4 ±0.6 Mepivacaine HCl 182 27.8 ± 0.2 Clindamycin Phosphate 209 36.5 ± 0.0Chloroquine Phosphate (0.10M) 179 37.4 ± 0.9 199 54.8 ± 0.2Phenylephrine HCl 183 54.1 ± 2.9 Moxifloxacin HCl 186 66.7 ± 1.0Piperacillin sodium salt 182 75.3 ± 1.6 Penicillin G sodium salt 17882.1 ± 3.6 Levetiracetam 176 103 ± 3  199 178 ± 2  Fosphenytoin disodiumsalt 188 119 ± 2  Ceftriaxone sodium salt 190 120 ± 2  Colistin sulfatesalt 203 138 ± 4  Cefoxitin sodium salt 194 166 ± 8  Aztreonam (0.02M)179 256 ± 4  Cidofovir hydrate (0.02M) 189 284 ± 5 

TABLE 38 Viscosities of Aqueous Solutions of REMICADE ® in the Presenceof 0.15M Viscosity-Lowering Agents (Unless Otherwise Indicated).[REMICADE ®], Agent mg/mL Viscosity, cP PB 176 432 ± 30 Creatinine 14437.1 ± 0.5 Procaine HCl 174 23.4 ± 0.2 Thiamine HCl 178 40.7 ± 0.3

Example 25. Viscosity-Lowering Effect of TPP and TPPAPMI, as a Functionof Concentration of Biosimilar AVASTIN®

Aqueous solutions of a commercially-obtained biosimilar AVASTIN® wereprepared as described in Example 1 above. The protein was formulated tocontain either 0.25 M phosphate buffer, 0.10 M thiamine pyrophosphate(TPP), or 0.10 M TPP-1-(3-aminopropyl)-2-methyl-1H-imidazole (TPPAPMI).

FIG. 8 depicts the viscosity of aqueous biosimilar AVASTIN® solutions asa function of mAb concentration with either phosphate buffer, TPP, orTPPAPMI. The viscosity of biosimilar AVASTIN® in phosphate bufferincreases exponentially within the tested protein concentration range.In the presence of TPP-containing excipients, the increase in viscosityis attenuated i.e. the viscosity gradient is reduced.

Example 26: Viscosity-Reducing Effect of a Viscosity-Lowering Agent,Thiamine HCl, as a Function of Concentration of Biosimilar SIMPONI ARIA®

Materials and Methods

SIMPONI ARIA® obtained commercially and containing pharmaceuticalexcipients (Histidine, Sorbitol, Polysorbate 80) was purified, bufferexchanged, concentrated, dried, reconstituted, and analyzed as describedin Example 1 above (using the extinction coefficient of 1.4 L/g·cm at280 nm). The protein was formulated to contain either 0.15 M phosphatebuffer or 0.15 M thiamine HCl.

Results

FIG. 9 depicts the viscosity of aqueous SIMPONI ARIA® solutions as afunction of mAb concentration with either phosphate buffer or thiamineHCl. The viscosity of SIMPONI ARIA® in phosphate buffer increasesexponentially within the tested protein concentration range. In thepresence of thiamine HCl, the increase in viscosity is attenuated i.e.the viscosity gradient is reduced.

Example 27. Viscosity-Lowering Effect of Thiamine HCl, as a Function ofConcentration of ENBREL®

Materials and Methods

ENBREL® obtained commercially and containing pharmaceutical excipients(Mannitol, Sucrose, Tromethamine) was purified, buffer exchanged,concentrated, dried, reconstituted, and analyzed as described in Example1 above (using the extinction coefficient of 0.96 L/g·cm at 280 nm). Theprotein was formulated to contain either 0.15 M phosphate buffer or 0.15M Thiamine HCl.

Results

Table 39 depicts the viscosity of aqueous ENBREL® solutions with eitherphosphate buffer or thiamine HCl. The addition of thiamine HCl reducesthe viscosity of ENBREL® up to about 2-fold.

TABLE 39 Viscosities of Aqueous Solutions of ENBREL ® in the Presence of0.15M PB or Thiamin HCl [ENBREL], 0.15M Thiamin mg/mL 0.15M PB HCl 271 ±0 1120 ± 26   626 ± 32 250 ± 3 439 ± 11 305 ± 7 212 ± 7 316 ± 11 141 ± 3

Example 28. Isotonic Solutions of Viscosity-Lowering Excipients Reducethe Viscosity of Concentrated Solutions of REMICADE®

Materials and Methods

Commercially-obtained REMICADE® containing pharmaceutical excipients(sucrose, Polysorbate 80, sodium phosphate buffer) was prepared as perinstructions in the prescribing information sheet. Subsequently, theaqueous drug product was purified, buffer exchanged, concentrated,dried, reconstituted, and analyzed as described in Example 1, exceptthat iosotonic amounts of charged hydrophobic compounds were added.

Results

As demonstrated in Table 40, isotonic amounts of both CSAA and CSAAPMIare capable of substantially reducing the viscosity of concentratedsolutions of REMICADE®, in some cases by up to about 10-fold.

TABLE 40 Viscosities of solutions of REMICADE ® in the presence ofisotonic (0.3 molal) viscosity-lowering excipients [REMICADE ®] Salt(mg/mL) Viscosity (cP) PB 171 432 ± 30 CSAAPMI 167 41.4 ± 0.7 PB 131 175± 15 CSAAPMI 124 16.4 ± 1.2 CSAA 128 25.8 ± 0.8

Unless expressly defined otherwise above, all technical and scientificterms used herein have the same meanings as commonly understood by oneof skill in the art. Those skilled in the art will recognize, or will beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the followingclaims.

We claim:
 1. A liquid pharmaceutical formulation for injectioncomprising: (i) an antibody; (ii) procaine; and (iii) a pharmaceuticallyacceptable solvent; wherein the liquid pharmaceutical formulation, whenin a volume suitable for injection, has an absolute viscosity of fromabout 1 cP to about 50 cP at 25° C. as measured using a cone and plateviscometer or a microfluidic viscometer; and the absolute viscosity ofthe liquid pharmaceutical formulation is less than an absolute viscosityof a control composition comprising the antibody and thepharmaceutically acceptable solvent but without the procaine; andwherein the absolute viscosity is an extrapolated zero-shear viscosity.2. The liquid pharmaceutical formulation of claim 1, wherein theantibody has a molecular weight of from about 120 kDa to about 250 kDa.3. The liquid pharmaceutical formulation of claim 1, comprising fromabout 174 mg/ml to about 230 mg/ml of the antibody.
 4. The liquidpharmaceutical formulation of claim 1, wherein the pharmaceuticallyacceptable solvent is aqueous.
 5. The liquid pharmaceutical formulationof claim 1, further comprising one or more pharmaceutically acceptableexcipients, the one or more pharmaceutically acceptable excipientscomprising a sugar, sugar alcohol, buffering agent, preservative,carrier, antioxidant, chelating agent, natural polymer, syntheticpolymer, cryoprotectant, lyoprotectant, surfactant, bulking agent,stabilizing agent, or any combination thereof.
 6. The liquidpharmaceutical formulation of claim 5, wherein the one or morepharmaceutically acceptable excipients comprise a polysorbate, poloxamer188, sodium lauryl sulfate, a polyol, poly(ethylene glycol), glycerol,propylene glycol, or a poly(vinyl alcohol).
 7. The liquid pharmaceuticalformulation of claim 1 in a unit-dose vial, multi-dose vial, cartridge,or pre-filled syringe.
 8. The liquid pharmaceutical formulation of claim1, wherein the liquid pharmaceutical formulation is isotonic to humanblood serum.
 9. The liquid pharmaceutical formulation of claim 1,wherein the absolute viscosity is measured at a shear rate of at leastabout 0.5 s⁻¹, when measured using a cone and plate viscometer.
 10. Theliquid pharmaceutical formulation of claim 1, wherein the absoluteviscosity is measured at a shear rate of at least about 1.0 s⁻¹, whenmeasured using a microfluidic viscometer.
 11. The liquid pharmaceuticalformulation of claim 1, wherein the antibody is a monoclonal antibody.12. The liquid pharmaceutical formulation of claim 5, wherein the sugaralcohol is sorbitol or mannitol.
 13. The liquid pharmaceuticalformulation of claim 1, wherein the liquid pharmaceutical formulation isreconstituted from a lyophilized composition.
 14. A lyophilizedcomposition comprising: (i) an antibody; (ii) procaine; and (iii) apharmaceutically acceptable excipient.
 15. The lyophilized compositionof claim 14, wherein, once reconstituted, the antibody has aconcentration of at least 100 mg/ml.
 16. A method of administering atherapeutically effective amount of an antibody to a subject, the methodcomprising subcutaneously or intramuscularly injecting the liquidpharmaceutical formulation of claim 1 into the subject.
 17. The methodof claim 16, wherein the injecting produces a primary irritation indexof less than 3 when evaluated using a Draize scoring system.
 18. Themethod of claim 16, wherein the injecting has an injection force that isat least 10% less than an injection force for a control composition, thecontrol composition comprising the antibody and the pharmaceuticallyacceptable solvent, but without the procaine, wherein the controlcomposition is administered in the same way as the liquid pharmaceuticalformulation.
 19. The method of claim 16, wherein the injecting isperformed with a needle between 27 and 31 gauge in diameter.
 20. Amethod of preparing the liquid pharmaceutical formulation of claim 1comprising the step of combining the antibody, the pharmaceuticallyacceptable solvent, and the procaine.
 21. The method of claim 16,wherein the injecting is performed with a heated syringe, a self-mixingsyringe, an auto-injector, a pre-filled syringe, or combinationsthereof.
 22. The method of claim 18, wherein the injecting has aninjection force that is at least 20% less than an injection force for acontrol composition, the control composition comprising the antibody andthe pharmaceutically acceptable solvent, but without the procaine,wherein the control composition is administered in the same way as theliquid pharmaceutical formulation.
 23. The method of claim 16, whereinthe liquid pharmaceutical formulation has a volume equal to or less thanabout 1.5 mL for the subcutaneous injecting, or equal to or less thanabout 3 mL for the intramuscular injecting.
 24. The method of claim 19,wherein the injecting has an injection force that is less than 30 N witha 27 gauge needle.